Induced exon inclusion in spinal muscle atrophy

ABSTRACT

The invention relates to the use of an antisense compound for inducing exon inclusion as a treatment for Spinal Muscle Atrophy (SMA). More particularly it relates to inducing inclusion of exon 7 to restore levels of Survival Motor Neuron (SMN) protein encoded by the Survival Motor Neuron (SMN) gene.

RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national stage filing ofInternational Application PCT/US2012/067475, filed Nov. 30, 2012, whichclaims priority to U.S. Provisional Patent Application 61/565,499 filedNov. 30, 2011. The contents of the aforementioned applications arehereby incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The invention relates to the use of an antisense compound for inducingexon inclusion as a treatment for Spinal Muscle Atrophy (SMA). Moreparticularly it relates to inducing inclusion of exon 7 to restorelevels of Survival Motor Neuron (SMN) protein encoded by the SurvivalMotor Neuron (SMN) gene.

BACKGROUND

Alternative splicing increases the coding potential of human genome byproducing multiple proteins from a single gene. It is also associatedwith a growing number of human diseases.

SMA is an often-fatal genetic disorder resulting from the loss of theSMN protein encoded by the Survival Motor Neuron SMN gene. The SMNgenes, SMN1 and SMN2, are located on chromosome 5 and SMA is caused bythe loss of SMN1 from both chromosomes. SMN2, while being almostidentical to SMN1, is less effective at making the SMN protein. Theseverity of SMA is affected by the efficiency at which SMN2, of whichthere are several copies, produces the SMN protein.

SMN1 encodes a ubiquitously expressed 38 kDa SMN protein that isnecessary for snRNP assembly, an essential process for cell survival. Anearly identical copy of the gene, SMN2, fails to compensate for theloss of SMN1 because of exon 7 skipping, producing an unstable truncatedprotein, SMNΔ7. SMN1 and SMN2 differ by a critical C to T substitutionat position 6 of exon 7 (C6U in transcript of SMN2). C6U does not changethe coding sequence, but is sufficient to cause exon 7 skipping in SMN2.

Current treatment for SMA consists of prevention and management of thesecondary effect of chronic motor unit loss. Currently, there are nodrug therapies available for the treatment or prevention of SMA.

Antisense technology, used mostly for RNA downregulation, recently hasbeen adapted to alter the splicing process. Effective agents that canalter splicing of SMN2 pre-mRNAs are likely to be usefultherapeutically.

SUMMARY OF THE INVENTION

The present application relates to methods of enhancing the level ofexon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in acell, comprising contacting the cell with an antisense oligonucleotideof sufficient length and complementarity to specifically hybridize to aregion within the SMN2 gene, such that the level of exon 7-containingSMN2 mRNA relative to exon-deleted SMN2 mRNA in the cell is enhanced,wherein the antisense oligonucleotide has at least one nucleoside thatis positively charged at physiological pH.

In one embodiment, the antisense oligonucleotide has at least oneinternucleoside linkage that exhibits a pKa between about 4.5 and about12. Preferably, the antisense oligonucleotide has an internucleosidelinkage containing both a basic nitrogen and an alkyl, aryl, or aralkylgroup. In other embodiments the antisense oligonucleotide comprises amorpholino.

In other embodiments the antisense oligonucleotide includes at least onenucleotide having a formula:

-   -   wherein Nu is a nucleobase;    -   R₁ is a moiety of the formula

-   -   q is 0, 1, 2, 3 or 4;    -   R₂ is selected from the group consisting of hydrogen, C₁-C₅        alkyl, and a formamidinyl moiety, and    -   R₃ is selected from the group consisting of hydrogen and C₁-C₅        alkyl, or    -   R₂ and R₃ are joined to form a 5-7 membered heterocyclic ring        optionally containing an oxygen hetero atom, where the ring may        be optionally substituted with a substituent selected from the        group consisting of C₁-C₅ alkyl, phenyl, halogen, and aralkyl;    -   R₄ is selected from the group consisting of null, hydrogen, a        C₁-C₆ alkyl and aralkyl;    -   R_(x) is selected from the group consisting of HO—, a        nucleotide, a cell penetrating peptide moiety, and piperazinyl;    -   R_(y) is selected from the group consisting of hydrogen, a C₁-C₆        alkyl, a nucleotide, a peptide moiety, an amino acid, a        formamidinyl moiety, and acyl; and,    -   R_(z) is selected from the group consisting of null, hydrogen, a        C₁-C₆ alkyl, and acyl;    -   and pharmaceutically acceptable salts thereof.        Preferably, Nu is selected from the group consisting of adenine,        guanine, thymine, uracil, cytosine, and hypoxanthine.

Other methods of enhancing the level of exon 7-containing SMN2 mRNArelative to exon-deleted SMN2 mRNA in a cell, comprise contacting thecell with an antisense oligonucleotide of sufficient length andcomplementarity to specifically hybridize to a region within the SMN2gene, such that the level of exon 7-containing SMN2 mRNA relative toexon-deleted SMN2 mRNA in the cell is enhanced, wherein the antisenseoligonucleotide has at least one nucleoside that has the formula:

-   -   wherein Rx, Ry, Rz, and Nu are as stated above.

In another embodiment, methods of enhancing the level of exon7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in a cellinclude contacting the cell with an antisense oligonucleotide ofsufficient length and complementarity to specifically hybridize to aregion within the SMN2 gene, such as a region within exon 7, intron 7,or exon 8 (or a region which spans a splice junction) of the SMN2 gene,thereby enhancing the level of exon 7-containing SMN2 mRNA relative toexon-deleted SMN2 mRNA in the cell. Preferably, the antisenseoligonucleotide comprises a sequence which is complementary to intron 7of the SMN2 gene or complementary to exon 8 of the SMN2 gene. Anotherembodiment relates to antisense oligonucleotides that further comprisesa peptide moiety which enhances cellular uptake.

In one embodiment, the antisense oligonucleotide is uncharged. Inadditional embodiments, the antisense oligonucleotide is charged. Forexample, one or more internucleotide linkages in the antisenseoligonucleotide may have an APN modification. The modifiedoligonucleotides contain nucleobases T, A, C, G, U or an analog thereof.Preferably the modified internucleotide linkage is derived from a T, Cor A subunit.

The invention also pertains to antisense oligonucleotides set forth inTable 1 having an APN modification and use thereof in the methods of theinvention.

Methods of treating spinal muscular atrophy (SMA) in a patient are alsowithin the scope of the invention. Such methods include administering tothe patient an antisense oligonucleotide comprising a nucleotidesequence of sufficient length and complementarity to specificallyhybridize to a region within the SMN2 gene, such that the level of exon7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in the cell isenhanced, wherein the antisense oligonucleotide has at least onenucleoside that is positively charged at physiological pH, therebytreating the patient.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 and 2: Preparation of the solid support for synthesis ofmorpholino oligomers.

FIGS. 3 and 4: The solid phase synthesis of morpholino oligomers.

FIG. 5: Spinal Muscular Atrophy (SMA) Sequences Tested. Oligonucleotidescontaining the sequences described above and tested in FIG. 6 contain aPMO backbone with an APN modification at the bold and underlined T bases(4 total modifications for each APN-containing oligo).

FIG. 6: Dose response curves from oligonucleotide-treated SpinalMuscular Atrophy patient fibroblasts. Intensity of the gel bandsrepresenting inclusion or exclusion of SMN2 exon 7 in GM03813 fibroblastcells (Coriell) were quantified with ImageQuant (GE). Exon 7 inclusionis reported as a percentage calculated from the ratio of the exon7-included band intensities divided by the sum of the intensities fromthe exon 7-included and -excluded bands. Each dot represents themean+/−1 standard deviation of two replicates at each concentration.Three independent experiments were combined to yield the above dataset.Percent inclusion analysis was performed in Microsoft Excel. Data pointsand curves were plotted in Graphpad Prism. The data shows that APNmodifications to an oligonucleotide enhances the potency of the compoundcompared to unmodified PMO containing the same sequence.

FIG. 7: RT-PCR of SMN2. RNA from GM03813 fibroblasts nucleofected withthe E8/4b APN-modified oligonucleotide at the indicated concentrationswere RT-PCR amplified as described in the Methods. Gels containing theresulting reactions were analyzed as described in the Methods and inFIG. 6.

FIG. 8: As is shown in FIG. 7 the invention provides for a method forenhancing the level of exon 7-containing SMN2 mRNA relative toexon-deleted SMN2 mRNA in a cell. This is shown schematically in thisfigure. The largest band on the gel in FIG. 7. corresponds to a cDNAcontaining Exons 6, 7, intron-7 and Exon 8. The middle band contains areverse transcription product containing Exons 6, 7, and Exon 8. Thelowest band corresponds to Exons 6 and 8. Thus, the treatment of cellswith the APN oligonucleotide increase the higher molecular weight bandscorresponding to the inclusion of Exon 7 in the cDNA. Since Exon 7includes a stop codon the protein product of both high molecular weightcDNAs will be the same.

FIG. 9: Exemplary structures of APN- and plus-related cationicmodifications. Shown are exemplary species of APN-related andplus-related cationic modifications. APN-related modifications includeAPN and mapT, and plus-related modifications include plusT, medaT, andetpipT. Although the exemplified modifications relate to thymine, anybase (e.g., thymine, cytosine, guanine, adenine) can be modified withthe APN-related and plus-related cationic modifications.

DETAILED DESCRIPTION

As used herein, “nucleobase” (Nu), “base pairing moiety” or “base” areused interchangeably to refer to a purine or pyrimidine base found innative DNA or RNA (uracil, thymine, adenine, cytosine, and guanine), aswell as analogs of the naturally occurring purines and pyrimidines, thatconfer improved properties, such as binding affinity to theoligonucleotide. Exemplary analogs include hypoxanthine (the basecomponent of the nucleoside inosine); 5-methyl cytosine;C5-propynyl-modified pyrimidines, 9-(aminoethoxy)phenoxazine (G-clamp)and the like.

Further examples of base pairing moieties include, but are not limitedto, uracil, thymine, adenine, cytosine, and guanine having theirrespective amino groups protected by acyl protecting groups,2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil,2,6-diaminopurine, azacytosine, pyrimidine analogs such aspseudoisocytosine and pseudouracil and other modified nucleobases suchas 8-substituted purines, xanthine, or hypoxanthine (the latter twobeing the natural degradation products). The modified nucleobasesdisclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al.Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao,Comprehensive Natural Products Chemistry, vol. 7, 313, are alsocontemplated.

Further examples of base pairing moieties include, but are not limitedto, expanded-size nucleobases in which one or more benzene rings hasbeen added. Nucleic base replacements described in the Glen Researchcatalog (www.glenresearch.com); Krueger A T et al, Acc. Chem. Res.,2007, 40, 141-150; Kool, E T, Acc. Chem. Res., 2002, 35, 936-943; BennerS. A., et al., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F. E., etal., Curr. Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I., Curr. Opin.Chem. Biol., 2006, 10, 622-627, are contemplated as useful for thesynthesis of the oligomers described herein. Some examples of theseexpanded-size nucleobases are shown below:

A nucleobase covalently linked to a ribose, sugar analog or morpholinocomprises a nucleoside. “Nucleotides” are composed of a nucleosidetogether with one phosphate group. The phosphate groups covalently linkadjacent nucleotides to one another to form an oligonucleotide. As usedherein, an “oligonucleotide” is a linear sequence of nucleotides, ornucleotide analogs, that allows the nucleobase to hybridize to a targetsequence in an RNA by Watson-Crick base pairing, to form anoligonucleotide:RNA heteroduplex within the target sequence. The terms“antisense oligonucleotide”, “antisense oligomer”, “oligomer” and“compound” may be used interchangeably to refer to an oligonucleotide.

A “morpholino oligomer” or “PMO” refers to an oligonucleotide having abackbone which supports a nucleobase capable of hydrogen bonding totypical polynucleotides, wherein the polymer lacks a pentose sugarbackbone moiety, but instead contains a morpholino ring. Thus, in a PMOa morpholino ring structure supports a base pairing moiety, to form asequence of base pairing moieties which is typically designed tohybridize to a selected antisense target in a cell or in a subject beingtreated. An exemplary “morpholino” oligomer comprises morpholino subunitstructures linked together by phosphoramidate or phosphorodiamidatelinkages, joining the morpholino nitrogen of one subunit to the 4′exocyclic carbon of an adjacent subunit, each subunit comprising apurine or pyrimidine nucleobase effective to bind, by base-specifichydrogen bonding, to a base in a polynucleotide. Morpholino oligomers(including antisense oligomers) are detailed, for example, in U.S. Pat.Nos. 5,698,685; 5,217,866; 5,142,047; 5,034,506; 5,166,315; 5,185,444;5,521,063; 5,506,337 and pending U.S. patent application Ser. Nos.12/271,036; 12/271,040; and PCT publication number WO/2009/064471 all ofwhich are incorporated herein by reference in their entirety.

Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the “internucleoside linkages” of theoligonucleotide. The naturally occurring internucleoside linkage of RNAand DNA is a 3′ to 5′ phosphodiester linkage. A “phosphoramidate” groupcomprises phosphorus having three attached oxygen atoms and one attachednitrogen atom, while a “phosphorodiamidate” group comprises phosphorushaving two attached oxygen atoms and two attached nitrogen atoms. In theuncharged or the cationic intersubunit linkages of the PMO and/or PMOXoligomers described herein, one nitrogen is always pendant to thebackbone chain. The second nitrogen, in a phosphorodiamidate linkage, istypically the ring nitrogen in a morpholino ring structure.

“PMOX” refers to phosphorodiamidate morpholino oligomers having aphosphorus atom with (i) a covalent bond to the nitrogen atom of amorpholino ring and (ii) a second covalent bond to the ring nitrogen ofa 4-aminopiperidin-1-yl (i.e. APN) or a derivative of4-aminopiperidin-1-yl. PMOX oligomers are disclosed in PCT applicationNo. PCT/US11/38459 (published as WO/2011/150408), herein incorporated byreference in its entirety. “PMOapn” or “APN” refers to a PMOX oligomerwhere a phosphorus atom is linked to a morpholino group and to the ringnitrogen of a 4-aminopiperidin-1-yl (i.e. APN).

As used herein, LNA refers to locked nucleic acid oligonucleotides.“LNA” are a member of a class of modifications called bridged nucleicacid (BNA). BNA is characterized by a covalent linkage that locks theconformation of the ribose ring in a C30-endo (northern) sugar pucker.For LNA, the bridge is composed of a methylene between the 2′-O and the4′-C positions. LNA enhances backbone preorganization and base stackingto increase hybridization and thermal stability.

An oligonucleotide “specifically hybridizes” to a target polynucleotideif the oligomer hybridizes to the target under physiological conditions,with a Tm substantially greater than 45° C., preferably at least 50° C.,and typically 60° C.-80° C. or higher. Such hybridization preferablycorresponds to stringent hybridization conditions. At a given ionicstrength and pH, the Tm is the temperature at which 50% of a targetsequence hybridizes to a complementary polynucleotide. Suchhybridization may occur with “near” or “substantial” complementarity ofthe antisense oligomer to the target sequence, as well as with exactcomplementarity.

A targeting sequence may have “near” or “substantial” complementarity tothe target sequence and still function for the purpose of the presentinvention, that is, still be “complementary.” Preferably, theoligonucleotide analog compounds employed in the present invention haveat most one mismatch with the target sequence out of 10 nucleotides, andpreferably at most one mismatch out of 20. Alternatively, the antisenseoligomers employed have at least 90% sequence homology, and preferablyat least 95% sequence homology, with the exemplary targeting sequencesas designated herein.

“An electron pair” refers to a valence pair of electrons that are notbonded or shared with other atoms.

The terms “cell penetrating peptide” (CPP) or “a peptide moiety whichenhances cellular uptake” are used interchangeably and refer to cationiccell penetrating peptides, also called “transport peptides”, “carrierpeptides”, or “peptide transduction domains”. The peptides, as shownherein, have the capability of inducing cell penetration within 30%,40%, 50%, 60%, 70%, 80%, 90% or 100% of cells of a given cell culturepopulation, including all integers in between, and allow macromoleculartranslocation within multiple tissues in vivo upon systemicadministration. In one embodiment, the cell-penetrating peptide may bean arginine-rich peptide transporter. In another embodiment, thecell-penetrating peptide may be Penetratin or the Tat peptide. Thesepeptides are well known in the art and are disclosed, for example in USPublication No. 2010-0016215 A1, incorporated by reference in itsentirety. A particularly preferred approach to conjugation of peptidesto antisense oligonucleotides can be found in PCT publicationWO2012/150960 which is incorporated by reference in its entirety. Apreferred embodiment of a peptide conjugated oligo utilizes glycine asthe linker between the CPP and the antisense oligonucleotide. Forexample, antisense oligonucleotides of the invention can be coupled toan arginine-rich peptide, such as (Arg)₆Gly (6 arginine and 1 glycinelinked to an oligonucleotide); e.g., a preferred peptide conjugated PMOconsists of R6-G-PMO.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated polynucleotide” or “isolated oligonucleotide,” asused herein, may refer to a polynucleotide that has been purified orremoved from the sequences that flank it in a naturally-occurring state,e.g., a DNA fragment that is removed from the sequences that areadjacent to the fragment in the genome. The term “isolating” as itrelates to cells refers to the purification of cells (e.g., fibroblasts,lymphoblasts) from a source subject (e.g., a subject with apolynucleotide repeat disease). In the context of mRNA or protein,“isolating” refers to the recovery of mRNA or protein from a source,e.g., cells.

As used herein, “sufficient length” refers to an antisenseoligonucleotide that is complementary to at least 8, more typically8-40, contiguous nucleobases in the RNA. An antisense oligonucleotide ofsufficient length has at least a minimal number of nucleotides to becapable of specifically hybridizing to the RNA. Preferably anoligonucleotide of sufficient length is from 10 to 40 nucleotides inlength, including oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39 and 40 nucleotides. In one embodiment, anoligonucleotide of sufficient length is from 10 to about 30 nucleotidesin length. In another embodiment, an oligonucleotide of sufficientlength is from 15 to about 25 nucleotides in length. In yet anotherembodiment, an oligonucleotide of sufficient length is from 20 to about30 nucleotides in length.

As used herein, the terms “contacting a cell”, “introducing” or“delivering” refers to delivery of the oligonucleotides of the inventioninto a cell by methods routine in the art, e.g., transfection (e.g.,liposome, calcium-phosphate, polyethyleneimine), electroporation (e.g.,nucleofection), microinjection).

As used herein, the term “quantifying”, “quantification” or otherrelated words refer to determining the quantity, mass, or concentrationin a unit volume, of a nucleic acid, polynucleotide, oligonucleotide,peptide, polypeptide, or protein.

As used herein, “treatment” of a subject (e.g. a mammal, such as ahuman) or a cell is any type of intervention used in an attempt to alterthe natural course of the individual or cell. Treatment includes, but isnot limited to, administration of a pharmaceutical composition, and maybe performed either prophylactically or subsequent to the initiation ofa pathologic event or contact with an etiologic agent.

Structural Features of the Oligonucleotides

As noted above, the substantially uncharged oligonucleotide may bemodified, in accordance with an aspect of the invention, to includecharged linkages, e.g., up to about 1 per every 2-5 uncharged linkages,such as about 4-5 per every 10 uncharged linkages. In certainembodiments, optimal improvement in antisense activity may be seen whenabout 25% of the backbone linkages are cationic. In certain embodiments,enhancement may be seen with a small number e.g., 10-20% cationiclinkages, or where the number of cationic linkages are in the range50-80%, such as about 60%.

In certain embodiments, the antisense compounds can be prepared bystepwise solid-phase synthesis, employing methods detailed in thereferences cited above, and below with respect to the synthesis ofoligonucleotides having a mixture or uncharged and cationic backbonelinkages. In some cases, it may be desirable to add additional chemicalmoieties to the antisense compound, e.g., to enhance pharmacokinetics orto facilitate capture or detection of the compound. Such a moiety may becovalently attached, according to standard synthetic methods. Forexample, addition of a polyethyleneglycol moiety or other hydrophilicpolymer, e.g., one having 1-100 monomeric subunits, may be useful inenhancing solubility.

A reporter moiety, such as fluorescein or a radiolabeled group, may beattached for purposes of detection. Alternatively, the reporter labelattached to the oligomer may be a ligand, such as an antigen or biotin,capable of binding a labeled antibody or streptavidin. In selecting amoiety for attachment or modification of an antisense compound, it isgenerally of course desirable to select chemical compounds of groupsthat are biocompatible and likely to be tolerated by a subject withoutundesirable side effects.

As noted above, certain of the antisense compounds can be constructed tocontain a selected number of cationic linkages interspersed withuncharged linkages of the type described above. The intersubunitlinkages, both uncharged and cationic, preferably arephosphorus-containing linkages, having the structure:

whereW is S or O, and is preferably O,X═R₁, NR¹¹R¹² or OR¹⁶,Y═O or NR¹⁷,

and each said linkage in the oligomer is selected from:

(a) uncharged linkage (a), where each of R¹¹, R¹², R¹⁶ and R¹⁷ isindependently selected from hydrogen and C₁-C₆ alkyl; or

(b1) cationic linkage (1), where R₁ is a moiety of the formula

q is 0, 1, 2, 3 or 4;

R₂ is selected from the group consisting of hydrogen, C₁-C₅ alkyl, and aformamidinyl moiety, and

R₃ is selected from the group consisting of hydrogen and C₁-C₅ alkyl, or

R₂ and R₃ are joined to form a 5-7 membered heterocyclic ring optionallycontaining an oxygen heteroatom, where the ring may be optionallysubstituted with a substituent selected from the group consisting ofC₁-C₅ alkyl, phenyl, halogen, and aralkyl;

R₄ is selected from the group consisting of null, hydrogen, C₁-C₆ alkyland aralkyl;

(b2) cationic linkage (b2), where X═NR¹¹R¹² and Y═O, and NR¹¹R¹²represents an optionally substituted piperazino group of the formula

where

each R is independently H or CH₃,

R¹⁴ is H, CH₃, or null, and

R¹³ is selected from H, C₁-C₆ alkyl, 5-7 membered substituted orunsubstituted aryl, heteroaryl or heterocylic ring containing up to 2heteroatoms selected from the groups consisting of N and O, C(═NH)NH₂,Z-L-NRR, Z-L-NHC(═NH)NH₂, Z-L-COOH, Z-L-SH, Z-L-PPh₃, Z-L-R²¹—R²²,cholate, and [C(O)CHR′NH]_(m)H, where: Z is C(O) or a direct bond, L isan optional linker up to 18 atoms in length, preferably up to 12 atoms,and more preferably up to 8 atoms in length, having bonds selected fromalkyl, alkoxy, and alkylamino, R′ is a side chain of a naturallyoccurring amino acid or a one- or two-carbon homolog thereof, m is 1 to6, preferably 1 to 4; R²¹ is a 5-7 membered aryl ring, and R²² is a 5-7membered heteroaryl ring containing up to 4 heteroatoms selected fromthe groups consisting of N and O;

(b3) cationic linkage (b3), where X═NR¹¹R¹² and Y═O, R¹¹═H or CH₃, andR¹²=LNR¹³R¹⁴R¹⁵, where L, R¹³, and R¹⁴ are as defined above, and R¹⁵ isH, C₁-C₆ alkyl, or C₁-C₆ (alkoxy)alkyl; and

(b4) cationic linkage (b4), where Y═NR¹⁷ and X═OR¹⁶, andR¹⁷=LNR¹³R¹⁴R¹⁵, where L, R¹³, R¹⁴ and R¹⁵ are as defined above, and R¹⁶is H or C₁-C₆ alkyl;

and at least one said linkage is selected from cationic linkages (b1),(b2), (b3) and (b4).

In certain embodiments, an oligomer may include at least two consecutivelinkages of type (a) (i.e. uncharged linkages). In further embodiments,at least 5% of the linkages in the oligomer are cationic linkages (i.e.type (b1), (b2), (b3) or (b4)); for example, 10% to 60%, and preferably20-50% linkages may be cationic linkages.

In one embodiment, at least one linkage is of the type (b1), where, q is1, R₂ and R₃ are hydrogen and R₄ is null.

In one embodiment, at least one linkage is of type (b2), where,preferably, each R is H, R¹⁴ is H, CH₃, or null, and R¹³ is selectedfrom H, C₁-C₆ alkyl, C(═NH)NH₂, and C(O)-L-NHC(═NH)NH₂. The latter twoembodiments of R¹³ provide a guanidinyl moiety, either attached directlyto the piperazine ring, or pendant to a linker group L, respectively.For ease of synthesis, the variable Z in R¹³ is preferably C(O)(carbonyl), as shown.

The linker group L, as noted above, contains bonds in its backboneselected from alkyl (e.g., —CH₂—CH₂—), alkoxy (—C—O—), and alkylamino(e.g., —CH₂—NH—), with the proviso that the terminal atoms in L (e.g.,those adjacent to carbonyl or nitrogen) are carbon atoms. Althoughbranched linkages (e.g., —CH₂—CHCH₃—) are possible, the linker ispreferably unbranched. In one embodiment, the linker is a hydrocarbonlinker. Such a linker may have the structure —(CH₂)_(n)—, where n is1-12, preferably 2-8, and more preferably 2-6.

The morpholino subunits (nucleotide) have the structure:

where Pi is a base-pairing moiety, and the linkages depicted aboveconnect the nitrogen atom of (i) to the 5′ carbon of an adjacentsubunit. The base-pairing moieties Pi may be the same or different, andare generally designed to provide a sequence which binds to a targetnucleic acid.

The use of embodiments of linkage types (b1), (b2) (b3) and (b4) aboveto link morpholino subunits may be illustrated graphically as follows:

Preferably, but not necessarily, all cationic linkages in the oligomerare of the same type; i.e. all of type (b1), all of type (b2), all oftype (b3) or all of type (b4).

In further embodiments, the cationic linkages are selected from linkages(b2′) and (b2″) as shown below, where (b2′) is referred to herein as a“Pip” linkage and (b2″) is referred to herein as a “GuX” linkage:

In the structures above, W is S or O, and is preferably O; each of R¹¹and R¹² is independently selected from hydrogen and C₁-C₆ alkyl, and ispreferably methyl or ethyl; and A represents hydrogen or C₁-C₆ alkyl onone or more carbon atoms in (b2′) and (b2″). Preferably, the ringcarbons in the piperazine ring are unsubstituted; however, they mayinclude non-interfering substituents, such as methyl. Preferably, atmost one or two carbon atoms is so substituted. In further embodiments,at least 10% of the linkages are of type (b2′) or (b2″); for example,10%-60% and preferably 20% to 50%, of the linkages may be of type (b2′)or (b2″).

In certain embodiments, the oligomer contains no linkages of the type(b2′) above. Alternatively, the oligomer contains no linkages of type(b2) where each R is H, R¹³ is H or CH₃, and R¹⁴ is H, CH₃, or null.

The morpholino subunits may also be linked by non-phosphorus-basedintersubunit linkages, as described further below, where at least onelinkage is modified with a pendant cationic group as described above.

Other oligonucleotide analog linkages which are uncharged in theirunmodified state but which could also bear a pendant amine substituentcould be used. For example, a 5′-nitrogen atom on a morpholino ringcould be employed in a sulfamide linkage or a urea linkage (wherephosphorus is replaced with carbon or sulfur, respectively) and modifiedin a manner analogous to the 5′-nitrogen atom in structure (b4) above.

Oligomers having any number of cationic linkages are provided, includingfully cationic-linked oligomers. Preferably, however, the oligomers arepartially charged, having, for example, 10%-80%. In preferredembodiments, about 10% to 60%, and preferably 20% to 50% of the linkagesare cationic.

In one embodiment, the cationic linkages are interspersed along thebackbone. The partially charged oligomers preferably contain at leasttwo consecutive uncharged linkages; that is, the oligomer preferablydoes not have a strictly alternating pattern along its entire length.

Also considered are oligomers having blocks of cationic linkages andblocks of uncharged linkages; for example, a central block of unchargedlinkages may be flanked by blocks of cationic linkages, or vice versa.In one embodiment, the oligomer has approximately equal-length 5′, 3′and center regions, and the percentage of cationic linkages in thecenter region is greater than about 50%, preferably greater than about70%.

Oligomers for use in antisense applications generally range in lengthfrom about 10 to about 40 subunits, more preferably about 10 to 30subunits, and typically 15-25 bases. For example, an oligomer of theinvention having 19-20 subunits, a useful length for an antisensecompound, may ideally have two to ten, e.g., four to eight, cationiclinkages, and the remainder uncharged linkages. An oligomer having 14-15subunits may ideally have two to seven, e.g., 3, 4, or 5, cationiclinkages and the remainder uncharged linkages.

Each morpholino ring structure supports a base pairing moiety, to form asequence of base pairing moieties which is typically designed tohybridize to a selected antisense target in a cell or in a subject beingtreated. The base pairing moiety may be a purine or pyrimidine found innative DNA or RNA (e.g., A, G, C, T or U) or an analog, such ashypoxanthine (the base component of the nucleoside inosine) or 5-methylcytosine.

As noted above, certain embodiments are directed to oligomers comprisingnovel intersubunit linkages, including PMO-X oligomers and those havingmodified terminal groups. In some embodiments, these oligomers havehigher affinity for DNA and RNA than do the corresponding unmodifiedoligomers and demonstrate improved cell delivery, potency, and/or tissuedistribution properties compared to oligomers having other intersubunitlinkages. In one embodiment, the oligomers comprise at least oneintersubunit linkage of type (B) as defined herein. The oligomers mayalso comprise one or more intersubunit linkages of type (A) as definedherein. The structural features and properties of the various linkagetypes and oligomers are described in more detail in the followingdiscussion. The synthesis of these and related oligomers is described inco-owned U.S. application Ser. No. 13/118,298, which is incorporated byreference in its entirety.

In preferred embodiments, the invention provides for an oligonucleotidehaving a sequence complementary to the target sequence which isassociated with a human disease, and comprises a sequence of nucleotideshaving a formula:

wherein Nu is a nucleobase;

R₁ is selected from the group consisting of R₁′ and R₁″ wherein R₁′ isdimethylamino and R₁″ is a moiety of the formula

wherein at least one R₁ is R₁″;

q is 0, 1, 2, 3 or 4;

R₂ is selected from the group consisting of hydrogen, C₁-C₅ alkyl, and aformamidinyl moiety, and

R₃ is selected from the group consisting of hydrogen and C₁-C₅ alkyl, or

R₂ and R₃ are joined to form a 5-7 membered heterocyclic ring optionallycontaining an oxygen hetero atom, where the ring may be optionallysubstituted with a substituent selected from the group consisting ofC₁-C₅ alkyl, phenyl, halogen, and aralkyl;

R₄ is selected from the group consisting of null, hydrogen, a C₁-C₆alkyl and aralkyl;

Rx is selected from the group consisting of HO—, a nucleotide, a cellpenetrating peptide moiety, and piperazinyl;

Ry is selected from the group consisting of hydrogen, a C₁-C₆ alkyl, anucleotide, a peptide moiety, an amino acid, a formamidinyl moiety, andacyl; and,

Rz is selected from the group consisting of an null, hydrogen, a C₁-C₆alkyl, and acyl; and pharmaceutically acceptable salts thereof.

Nu may be selected from the group consisting of adenine, guanine,thymine, uracil, cytosine, and hypoxanthine. More preferably Nu isthymine or uracil.

About 50-90% of the R₁ groups are dimethylamino (i.e., R₁′). Most,preferably about 66% (two thirds) of the R₁ groups are dimethylamino.

R₁ may be selected from the group consisting of

Preferably, at least one nucleotide of the oligonucleotide has theformula:

wherein Rx, Ry, Rz, and Nu are as stated above. Most preferably, Nu isthymine or uracil.

Although thymine (T) is the preferred base pairing moiety (Nu or Pi)containing the chemical modifications described above, any base subunitknown to a person of skill in the art can be used as the base pairingmoiety.

Antisense Oligonucleotides

The invention provides for the use of an antisense oligonucleotide thatcomprises a sequence selected from the group set forth in Table 1.Preferably, the antisense oligonucleotide comprises a sequence selectedfrom the group consisting of 14-mer-APN, E-8/4a-APN and E8/4b-APN.Additional antisense oligonucleotides that can be used in accordancewith the present invention include those described in the followingpatents and patent publications, the contents of which are incorporatedherein by reference: WO2007/002390 WO2010/120820 WO2010/148249 U.S. Pat.No. 7,838,657 US 2011/0269820

The invention further relates to the antisense oligonucleotides setforth in Table 1 having an APN modification or APN derivative.Particular antisense oligonucleotides useful in the methods describedherein include 14-mer-APN, E-8/4a-APN and E8/4b-APN.

As used herein, the term “antisense oligonucleotide” refers to a nucleicacid (in preferred embodiments, an RNA) (or analog thereof), havingsufficient sequence complementarity to a target RNA (i.e., the RNA forwhich splice site selection is modulated) to block a region of a targetRNA (e.g., pre-mRNA) in an effective manner. In exemplary embodiments ofthe instant invention, such blocking of SMN2 pre-mRNA serves to modulatesplicing, either by masking a binding site for a native protein thatwould otherwise modulate splicing and/or by altering the structure ofthe targeted RNA. In preferred embodiments of the instant invention, thetarget RNA is target pre-mRNA (e.g., SMN2 pre-mRNA). An antisenseoligonucleotide having a sufficient sequence complementarity to a targetRNA sequence to modulate splicing of the target RNA means that theantisense agent has a sequence sufficient to trigger the masking of abinding site for a native protein that would otherwise modulate splicingand/or alters the three-dimensional structure of the targeted RNA.Likewise, an oligonucleotide reagent having a sufficient sequencecomplementary to a target RNA sequence to modulate splicing of thetarget RNA means that the oligonucleotide reagent has a sequencesufficient to trigger the masking of a binding site for a native proteinthat would otherwise modulate splicing and/or alters thethree-dimensional structure of the targeted RNA.

In some embodiments, the antisense oligonucleotide is uncharged. Inadditional embodiments, the antisense oligonucleotide is charged.

In some embodiments, the antisense oligonucleotide may be a “morpholinooligomer,” “PMO,” “PMOX,” “PPMO,” or “PMO+”. Furthermore, the antisenseoligonucleotide, e.g., PMO, may be modified in any manner known in theart. One or more internucleotide linkages in the antisenseoligonucleotide may be modified. For example, one or moreinternucleotide linkages in the antisense oligonucleotide may have acationic modification. The cationic modification may be an APNmodification. Preferably the modified internucleotide linkages arederived from a T, C or A subunit. For example, in one embodiment, thePMO may comprise a cationic modification. The PMO may be an APN modifiedPMO, which may be referred to as a “PMOapn” or “APN.”

As used herein, the term “SMA” refers to spinal muscular atrophy, ahuman autosomal recessive disease that is often characterized byunderexpression of SMN protein in affected individuals.

As used herein, the term “target” refers to a RNA region, andspecifically, to a region identified by the SMN2 gene. In a particularembodiment the target region is a region of the mRNA of the SMN2 intron7 region which is responsible for the deletion of exon 7 and isassociated with SMN. In another embodiment the target region is a regionof the mRNA of SMN2 exon 8.

The term “target sequence” refers to a portion of the target RNA againstwhich the oligonucleotide analog is directed, that is, the sequence towhich the oligonucleotide analog will hybridize by Watson-Crick basepairing of a complementary sequence.

The term “targeting sequence” is the sequence in the oligonucleotideanalog that is complementary (meaning, in addition, substantiallycomplementary) to the target sequence in the RNA genome. The entiresequence, or only a portion, of the antisense oligonucleotide may becomplementary to the target sequence. For example, in an antisenseoligonucleotide having 20 bases, only 12-14 may be targeting sequences.Typically, the targeting sequence is formed of contiguous bases in theoligonucleotide, but may alternatively be formed of non-contiguoussequences that when placed together, e.g., from opposite ends of theoligonucleotide, constitute sequence that spans the target sequence.

In general, oligonucleotide reagents containing nucleotide sequencesperfectly complementary to a portion of the target RNA are preferred forblocking of the target RNA. However, 100% sequence complementaritybetween the oligonucleotide reagent and the target RNA is not requiredto practice the present invention. Thus, the invention may toleratesequence variations that might be expected due to genetic mutation,strain polymorphism, or evolutionary divergence. For example,oligonucleotide reagent sequences with insertions, deletions, and singlepoint mutations relative to the target sequence may also be effectivefor inhibition. Alternatively, oligonucleotide reagent sequences withnucleotide analog substitutions or insertions can be effective forblocking.

Greater than 70% sequence identity (or complementarity), e.g., 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% oreven 100% sequence identity, between the oligonucleotide reagent and thetarget RNA, e.g., target pre-mRNA, is preferred. In addition, variantsof the oligonucleotide sequences set forth in Table 1 which retain thefunction of same can be used in the methods of the invention. Forexample, a series of mutants may be tested for their ability to inhibitalternative splicing. In one embodiment, such variant sequences are atleast about 95% identical in sequence to a sequence set forth in Table 1over the entire length of the same. In another embodiment, such variantsequences are at least about 90% identical in the sequence over theentire length of the same.

Splice forms and expression levels of surveyed RNAs and proteins may beassessed by any of a wide variety of well known methods for detectingsplice forms and/or expression of a transcribed nucleic acid or protein.Non-limiting examples of such methods include RT-PCR of spliced forms ofRNA followed by size separation of PCR products, nucleic acidhybridization methods e.g., Northern blots and/or use of nucleic acidarrays; nucleic acid amplification methods; immunological methods fordetection of proteins; protein purification methods; and proteinfunction or activity assays.

RNA expression levels can be assessed by preparing mRNA/cDNA (i.e. atranscribed polynucleotide) from a cell, tissue or organism, and byhybridizing the mRNA/cDNA with a reference polynucleotide which is acomplement of the assayed nucleic acid, or a fragment thereof. cDNA can,optionally, be amplified using any of a variety of polymerase chainreaction or in vitro transcription methods prior to hybridization withthe complementary polynucleotide; preferably, it is not amplified.Expression of one or more transcripts can also be detected usingquantitative PCR to assess the level of expression of the transcript(s).

Methods of the Invention

In one aspect, the invention provides for a method for enhancing thelevel of exon 7-containing SMN2 mRNA relative to exon-7 deleted SMN2mRNA in a cell, comprising contacting the cell with an antisenseoligonucleotide of sufficient length and complementarity to specificallyhybridize to a region within the SMN2 gene, such that the level of exon7-containing SMN2 mRNA relative to exon-7 deleted SMN2 mRNA in the cellis enhanced, wherein the antisense oligonucleotide has at least oneinternucleoside linkage that is positively charged at physiological pH.

Optionally, the antisense oligonucleotide may have internucleosidelinkage with both a basic nitrogen and an alkyl, aryl, or aralkyl group.Preferably, the antisense oligonucleotide comprises a morpholino.

Moreover, the invention provides that the antisense oligonucleotide hasat least one internucleoside linkage that is positively charged atphysiological pH. The invention also provides that the antisenseoligonucleotide has at least one internucleoside linkage that exhibits apKa between 5.5 and 12.

While not being bound by theory, it is believed that the positivelycharged APN group or APN derivatives, in the PMOX oligomer facilitatesbinding to the negatively charged phosphates in target nucleotide. Thus,the formation of a hetroduplex between mutant RNA and the PMOX oligomermay be held together by an ionic attractive force, as well as byWatson-Crick base pairing.

Effective delivery of the antisense oligomer to the target nucleic acidis an important aspect of treatment. Routes of antisense oligomerdelivery include, but are not limited to, various systemic routes,including oral and parenteral routes, e.g., intravenous, subcutaneous,intraperitoneal, and intramuscular, as well as inhalation, transdermaland topical delivery. The appropriate route may be determined by one ofskill in the art, as appropriate to the condition of the subject undertreatment. Vascular or extravascular circulation, the blood or lymphsystem, and the cerebrospinal fluid are some non-limiting sites wherethe RNA may be introduced.

The antisense oligonucleotides of the invention can be delivered to thenervous system of a subject by any art-recognized method. For example,peripheral blood injection of the antisense oligonucleotides of theinvention can be used to deliver said reagents to peripheral neurons viadiffusive and/or active means. In one embodiment, the antisenseoligonucleotides may be delivered to the brain of the subject. Forexample, the antisense oligonucleotides may be delivered byintracerebroventriclar (ICV) injection. Alternatively, the antisenseoligonucleotides of the invention can be modified to promote crossing ofthe blood-brain-barrier (BBB) to achieve delivery of said reagents toneuronal cells of the central nervous system (CNS). Specific recentadvancements in antisense oligonucleotide technology and deliverystrategies have broadened the scope of antisense oligonucleotide usagefor neuronal disorders (Forte, A., et al. 2005. Curr. Drug Targets6:21-29; Jaeger, L. B., and W. A. Banks. 2005. Methods Mol. Med.106:237-251; Vinogradov, S. V., et al. 2004. Bioconjug. Chem. 5:50-60;the preceding are incorporated herein in their entirety by reference).For example, the antisense oligonucleotides of the invention can begenerated as peptide nucleic acid (PNA) compounds. PNA reagents haveeach been identified to cross the BBB (Jaeger, L. B., and W. A. Banks.2005. Methods Mol. Med. 106:237-251). Treatment of a subject with, e.g.,a vasoactive agent, has also been described to promote transport acrossthe BBB (Id). Tethering of the antisense oligonucleotides of theinvention to agents that are actively transported across the BBB mayalso be used as a delivery mechanism.

In certain embodiments, the antisense oligonucleotides of the inventioncan be delivered by transdermal methods (e.g., via incorporation of theantisense oligonucleotides into, e.g., emulsions, with such antisenseoligonucleotides optionally packaged into liposomes). Such transdermaland emulsion/liposome-mediated methods of delivery are described fordelivery of antisense oligonucleotides in the art, e.g., in U.S. Pat.No. 6,965,025, the contents of which are incorporated in their entiretyby reference herein.

The antisense oligonucleotides of the invention may also be deliveredvia an implantable device. Design of such a device is an art-recognizedprocess, with, e.g., synthetic implant design described in, e.g., U.S.Pat. No. 6,969,400, the contents of which are incorporated in theirentirety by reference herein.

Antisense oligonucleotides can be introduced into cells usingart-recognized techniques (e.g., transfection, electroporation, fusion,liposomes, colloidal polymeric particles and viral and non-viral vectorsas well as other means known in the art). The method of deliveryselected will depend at least on the cells to be treated and thelocation of the cells and will be apparent to the skilled artisan. Forinstance, localization can be achieved by liposomes with specificmarkers on the surface to direct the liposome, direct injection intotissue containing target cells, specific receptor mediated uptake, viralvectors, or the like.

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, inhibit annealing of single strands,stabilize the single strands, or otherwise increase expression of thetarget gene.

As known in the art, antisense oligonucleotides may be delivered using,e.g., methods involving liposome-mediated uptake, lipid conjugates,polylysine-mediated uptake, nanoparticle-mediated uptake, andreceptor-mediated endocytosis, as well as additional non-endocytic modesof delivery, such as microinjection, permeabilization (e.g.,streptolysin-O permeabilization, anionic peptide permeabilization),electroporation, and various non-invasive non-endocytic methods ofdelivery that are known in the art (refer to Dokka and Rojanasakul,Advanced Drug Delivery Reviews 44, 35-49, incorporated in its entiretyherein by reference).

The antisense oligonucleotides may be administered in any convenientvehicle or carrier which is physiologically and/or pharmaceuticallyacceptable. Such a composition may include any of a variety of standardpharmaceutically acceptable carriers employed by those of ordinary skillin the art. Examples include, but are not limited to, saline, phosphatebuffered saline (PBS), water, aqueous ethanol, emulsions, such asoil/water emulsions or triglyceride emulsions, tablets and capsules. Thechoice of suitable physiologically acceptable carrier will varydependent upon the chosen mode of administration. “Pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The compounds (e.g., antisense oligonucleotides) of the presentinvention may generally be utilized as the free acid or free base.Alternatively, the compounds of this invention may be used in the formof acid or base addition salts. Acid addition salts of the free aminocompounds of the present invention may be prepared by methods well knownin the art, and may be formed from organic and inorganic acids. Suitableorganic acids include maleic, fumaric, benzoic, ascorbic, succinic,methanesulfonic, acetic, trifluoroacetic, oxalic, propionic, tartaric,salicylic, citric, gluconic, lactic, mandelic, cinnamic, aspartic,stearic, palmitic, glycolic, glutamic, and benzenesulfonic acids.

Suitable inorganic acids include hydrochloric, hydrobromic, sulfuric,phosphoric, and nitric acids. Base addition salts included those saltsthat form with the carboxylate anion and include salts formed withorganic and inorganic cations such as those chosen from the alkali andalkaline earth metals (for example, lithium, sodium, potassium,magnesium, barium and calcium), as well as the ammonium ion andsubstituted derivatives thereof (for example, dibenzylammonium,benzylammonium, 2-hydroxyethylammonium, and the like). Thus, the term“pharmaceutically acceptable salt” is intended to encompass any and allacceptable salt forms.

In addition, prodrugs are also included within the context of thisinvention. Prodrugs are any covalently bonded carriers that release acompound in vivo when such prodrug is administered to a patient.Prodrugs are generally prepared by modifying functional groups in a waysuch that the modification is cleaved, either by routine manipulation orin vivo, yielding the parent compound. Prodrugs include, for example,compounds of this invention wherein hydroxy, amine or sulfhydryl groupsare bonded to any group that, when administered to a patient, cleaves toform the hydroxy, amine or sulfhydryl groups. Thus, representativeexamples of prodrugs include (but are not limited to) acetate, formateand benzoate derivatives of alcohol and amine functional groups of theantisense oligonucleotides of the invention. Further, in the case of acarboxylic acid (—COOH), esters may be employed, such as methyl esters,ethyl esters, and the like.

In some instances, liposomes may be employed to facilitate uptake of theantisense oligonucleotide into cells. (See, e.g., Williams, S. A.,Leukemia 10(12):1980-1989, 1996; Lappalainen et al., Antiviral Res.23:119, 1994; Uhlmann et al., antisense oligonucleotides: a newtherapeutic principle, Chemical Reviews, Volume 90, No. 4, 25 pages544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers inBiology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels mayalso be used as vehicles for antisense oligomer administration, forexample, as described in WO 93/01286. Alternatively, theoligonucleotides may be administered in microspheres or microparticles.(See, e.g., Wu, G. Y. and Wu, C. H., J. Biol. Chem. 262:4429-4432, 301987). Alternatively, the use of gas-filled microbubbles complexed withthe antisense oligomers can enhance delivery to target tissues, asdescribed in U.S. Pat. No. 6,245,747. Sustained release compositions mayalso be used. These may include semipermeable polymeric matrices in theform of shaped articles such as films or microcapsules.

In one embodiment, the antisense oligonucleotide is administered to amammalian subject, e.g., human or domestic animal, exhibiting thesymptoms of a polynucleotide-repeat disorder, in a suitablepharmaceutical carrier. In one aspect of the method, the subject is ahuman subject, e.g., a patient diagnosed as having SMA. The patient'scondition may also dictate prophylactic administration of an antisenseoligonucleotides of the invention, e.g., in the case of a patient who(1) is immunocompromised; (2) is a burn victim; (3) has an indwellingcatheter; or (4) is about to undergo or has recently undergone surgery.In one preferred embodiment, the antisense oligonucleotide is containedin a pharmaceutically acceptable carrier, and is delivered orally. Inanother preferred embodiment, the oligomer is contained in apharmaceutically acceptable carrier, and is delivered intravenously(i.v.).

In one embodiment, the antisense compound is administered in an amountand manner effective to result in a peak blood concentration of at least200-400 nM antisense oligonucleotide. Typically, one or more doses ofantisense oligomer are administered, generally at regular intervals, fora period of about one to two weeks. Preferred doses for oraladministration are from about 1-1000 mg oligomer per 70 kg. In somecases, doses of greater than 1000 mg oligomer/patient may be necessary.For i.v. administration, preferred doses are from about 0.5 mg to 1000mg oligomer per 70 kg. The antisense oligomer may be administered atregular intervals for a short time period, e.g., daily for two weeks orless; once every two days; once every three days; once every 3 to 7days; once every 3 to 10 days; once every 7 to 10 days; once every twoweeks; once monthly. However, in some cases the oligomer is administeredintermittently over a longer period of time, e.g., for several weeks,months or years. For example, the antisense oligomer may be administeredonce every two, three, four, five, six, seven, eight, nine, ten, elevenor twelve months. In addition, the antisense oligomer may beadministered once every one, two, three, four or five years.Administration may be followed by, or concurrent with, administration ofan antibiotic or other therapeutic treatment. The treatment regimen maybe adjusted (dose, frequency, route, etc.) as indicated, based on theresults of immunoassays, other biochemical tests and physiologicalexamination of the subject under treatment.

An effective in vivo treatment regimen using the antisenseoligonucleotides of the invention may vary according to the duration,dose, frequency and route of administration, as well as the condition ofthe subject under treatment (i.e., prophylactic administration versusadministration in response to localized or systemic infection).Accordingly, such in vivo therapy will often require monitoring by testsappropriate to the particular type of disorder under treatment, andcorresponding adjustments in the dose or treatment regimen, in order toachieve an optimal therapeutic outcome.

Treatment may be monitored, e.g., by general indicators of disease knownin the art. The efficacy of an in vivo administered antisenseoligonucleotide of the invention may be determined from biologicalsamples (tissue, blood, urine etc.) taken from a subject prior to,during and subsequent to administration of the antisenseoligonucleotide. Assays of such samples include (1) monitoring thepresence or absence of heteroduplex formation with target and non-targetsequences, using procedures known to those skilled in the art, e.g., anelectrophoretic gel mobility assay; (2) monitoring the amount of amutant mRNA in relation to a reference normal mRNA or protein asdetermined by standard techniques such as RT-PCR, Northern blotting,ELISA or Western blotting.

In some embodiments, the antisense oligonucleotide is actively taken upby mammalian cells. In further embodiments, the antisenseoligonucleotide may be conjugated to a transport moiety (e.g., transportpeptide) as described herein to facilitate such uptake.

Methods of Treatment

The invention also relates to methods of increasing expression of exon7-containing SMN2 mRNA or protein using the antisense oligonucleotidesof the present invention for therapeutic purposes (e.g., treatingsubjects with SMA). Accordingly, in one embodiment, the presentinvention provides methods of treating an individual afflicted with SMA.

In one embodiment, cells from a subject having SMA are contacted with anantisense oligonucleotide of the invention to increase expression ofexon 7-containing SMN2 mRNA or protein. Exemplary antisense sequencesand compositions, such as 14-mer-APN, E-8/4a-APN and E8/4b-APN, aredisclosed in Table 1.

In one embodiment, cells from a subject having spinal muscular atrophyare contacted with an oligonucleotide reagent of the invention toinhibit splicing of the SMN2 exon 7. Exemplary oligonucleotide reagentsinclude sequences complementary to intron 7 target sequence or exon 8target sequence and variants thereof (e.g., as shown herein). In anotherembodiment, cells from a subject having another disorder that wouldbenefit from inhibition of alternative splicing are contacted with anoligonucleotide reagent of the invention. Target sequences related tothe target sequences disclosed herein are present in human intronicsequences. For example, there is a sequence partially homologous to anintron 7 sequence located in intron 10 of human CFTR.

Such agents can also be used in treatment of diseases associated withhigh susceptibility to oxidative stress such as exposure to Paraquat andinduced Parkinson's disease, as well as amyotrophic lateral sclerosis(ALS), another neurological disease characterized by low levels of SMNprotein (Veldink, J. H., et al. 2005 Neurology 65(6):820-5).

The antisense oligonucleotides of the invention can be administered tosubjects to treat (prophylactically or therapeutically) SMA. Inconjunction with such treatment, pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) may be considered. Differencesin metabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a therapeutic agent as wellas tailoring the dosage and/or therapeutic regimen of treatment with atherapeutic agent.

Comparison of APN Modified Oligonucleotides and UnmodifiedOligonucleotides

The oligonucleotides of some embodiments of the invention were tested todetermine whether an APN modified oligonucleotide would enhance SMN2Exon 7 inclusion as compared to the unmodified oligonucleotide. The APNmodified oligomer referred to as 14-mer-APN (T TTC ATA ATG CTG G)contains APN modifications on the T bases shown in bold (SEQ ID NO:21).The N1 (A TTC ACT TTC ATA ATG CTG G) and the 14mer oligomer (T TTC ATAATG CTG G) do not contain APN modifications (SEQ ID NOs: 1 and 19,respectively). Similarly, the oligomers referred to as E8/4a-APN(C TAGTAT TTC CTG CAA ATG AG) and E8/4b-APN(C CAG CAT TTC CTG CAA ATG AG)contain APN modifications on the T bases shown in bold (SEQ ID NOs: 43and 54, respectively); the corresponding oligomers referred to as E8/4a(C TAG TAT TTC CTG CAA ATG AG) and E8/4b (C CAG CAT TTC CTG CAA ATG AG)do not contain APN modifications (SEQ ID NOs: 42 and 53, respectively).See FIG. 5 and Table 1.

It was determined that the addition of APN modifications to anoligonucleotide enhances the potency of the compound compared tounmodified PMO containing the same sequence (See FIG. 6). Specifically,14 mer (11/15) APN was about an order of magnitude more potent than 14mer (11/15) without the APN linkage. Likewise E8/4a-APN (11/15) andE8/4b-APN (11/15) were more potent than E8/4a (11/15) and E8/4b (11/15)respectively (See Examples 24 and 25 and FIGS. 6 and 7).

The Preparation of PMO-X with Basic Nitrogen Internucleoside Linkers

Morpholino subunits, the modified intersubunit linkages and oligomerscomprising the same can be prepared as described in the examples and inU.S. Pat. Nos. 5,185,444, and 7,943,762 which are hereby incorporated byreference in their entirety. The morpholino subunits can be preparedaccording to the following general Reaction Scheme I.

Referring to Reaction Scheme 1, wherein B represents a base pairingmoiety and PG represents a protecting group, the morpholino subunits maybe prepared from the corresponding ribonucleoside (1) as shown. Themorpholino subunit (2) may be optionally protected by reaction with asuitable protecting group precursor, for example trityl chloride. The 3′protecting group is generally removed during solid-state oligomersynthesis as described in more detail below. The base pairing moiety maybe suitable protected for sold phase oligomer synthesis. Suitableprotecting groups include benzoyl for adenine and cytosine, phenylacetylfor guanine, and pivaloyloxymethyl for hypoxanthine (I). Thepivaloyloxymethyl group can be introduced onto the N1 position of thehypoxanthine heterocyclic base. Although an unprotected hypoxanthinesubunit, may be employed, yields in activation reactions are farsuperior when the base is protected. Other suitable protecting groupsinclude those disclosed in co-pending U.S. application Ser. No.12/271,040, which is hereby incorporated by reference in its entirety.

Reaction of 3 with the activated phosphorous compound 4, results inmorpholino subunits having the desired linkage moiety 5. Compounds ofstructure 4 can be prepared using any number of methods known to thoseof skill in the art. For example, such compounds may be prepared byreaction of the corresponding amine and phosphorous oxychloride. In thisregard, the amine starting material can be prepared using any methodknown in the art, for example those methods described in the Examplesand in U.S. Pat. No. 7,943,762.

Compounds of structure 5 can be used in solid-phase automated oligomersynthesis for preparation of oligomers comprising the intersubunitlinkages. Such methods are well known in the art. Briefly, a compound ofstructure 5 may be modified at the 5′ end to contain a linker to a solidsupport. For example, compound 5 may be linked to a solid support by alinker comprising L¹¹ and L¹⁵. An exemplary method is demonstrated inFIGS. 1 and 2. Once supported, the protecting group (e.g., trityl) isremoved and the free amine is reacted with an activated phosphorousmoiety of a second compound of structure 5. This sequence is repeateduntil the desired length of oligo is obtained. The protecting group inthe terminal 5′ end may either be removed or left on if a5′-modification is desired. The oligo can be removed from the solidsupport using any number of methods, for example treatment with DTTfollowed by ammonium hydroxide as depicted in FIGS. 3 and 4.

The preparation of modified morpholino subunits and morpholino oligomersare described in more detail in the Examples. The morpholino oligomerscontaining any number of modified linkages may be prepared using methodsdescribed herein, methods known in the art and/or described by referenceherein. Also described in the examples are global modifications ofmorpholino oligomers prepared as previously described (see e.g., PCTpublication WO2008036127).

The term “protecting group” refers to chemical moieties that block someor all reactive moieties of a compound and prevent such moieties fromparticipating in chemical reactions until the protective group isremoved, for example, those moieties listed and described in T. W.Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd ed.John Wiley & Sons (1999). It may be advantageous, where differentprotecting groups are employed, that each (different) protective groupbe removable by a different means. Protective groups that are cleavedunder totally disparate reaction conditions allow differential removalof such protecting groups. For example, protective groups can be removedby acid, base, and hydrogenolysis. Groups such as trityl,dimethoxytrityl, acetal and tert-butyldimethylsilyl are acid labile andmay be used to protect carboxy and hydroxy reactive moieties in thepresence of amino groups protected with Cbz groups, which are removableby hydrogenolysis, and Fmoc groups, which are base labile. Carboxylicacid moieties may be blocked with base labile groups such as, withoutlimitation, methyl, or ethyl, and hydroxy reactive moieties may beblocked with base labile groups such as acetyl in the presence of aminesblocked with acid labile groups such as tert-butyl carbamate or withcarbamates that are both acid and base stable but hydrolyticallyremovable.

Carboxylic acid and hydroxyl reactive moieties may also be blocked withhydrolytically removable protective groups such as the benzyl group,while amine groups may be blocked with base labile groups such as Fmoc.A particularly useful amine protecting group for the synthesis ofcompounds of Formula (I) is the trifluoroacetamide. Carboxylic acidreactive moieties may be blocked with oxidatively-removable protectivegroups such as 2,4-dimethoxybenzyl, while co-existing amino groups maybe blocked with fluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- andbase-protecting groups since the former are stable and can besubsequently removed by metal or pi-acid catalysts. For example, anallyl-blocked carboxylic acid can be deprotected with apalladium(0)-catalyzed reaction in the presence of acid labile t-butylcarbamate or base-labile acetate amine protecting groups. Yet anotherform of protecting group is a resin to which a compound or intermediatemay be attached. As long as the residue is attached to the resin, thatfunctional group is blocked and cannot react. Once released from theresin, the functional group is available to react.

Typical blocking/protecting groups are known in the art and include, butare not limited to the following moieties:

Unless otherwise noted, all chemicals were obtained fromSigma-Aldrich-Fluka. Benzoyl adenosine, benzoyl cytidine, andphenylacetyl guanosine were obtained from Carbosynth Limited, UK.

Synthesis of PMO, PMO+, PPMO, and PMO-X containing further linkagemodifications as described herein was done using methods known in theart and described in pending U.S. application Ser. Nos. 12/271,036 and12/271,040 and PCT publication number WO/2009/064471, which are herebyincorporated by reference in their entirety.

PMO with a 3′ trityl modification are synthesized essentially asdescribed in PCT publication number WO/2009/064471 with the exceptionthat the detritylation step is omitted.

Procedure a for the Preparation of Activated Subunits:

To a stirred solution of 6 (1 eq) in dichloromethane was added POCl₃(1.1 eq), followed by diisopropylethylamine (3 eq) at 0° C., cooled byan ice-bath. After 15 minutes, the ice-bath was removed and the solutionwas allowed to warm to room temperature for one hour. Upon reactioncompletion, the reaction solution was diluted with dichloromethane,washed with 10% aqueous citric acid three times. After drying overMgSO₄, the organic layer was passed through a plug of silica gel andconcentrated in vacuo. The resulting phosphoroamidodichloride (4) wasused directly for the next step without further purification.

To a solution of the phosphoroamidodichloride (4) (1 eq), 2,6-lutidine(1 eq) in dichloromethane was added Mo(Tr)T (7) (0.5 eq)/dichloromethanesolution, followed by N-methylimidazole (0.2 eq). The reaction stirredat room temperature overnight. Upon reaction completion, the reactionsolution was diluted with dichloromethane, and washed with 10% aqueouscitric acid three times. After drying over MgSO₄, the organic layer wasfiltered, then concentrated. The product (8) was purified by silica gelchromatography (eluting with a gradient of ethyl acetate/hexanes), andthen stored at −20° C. The structure was confirmed by LCMS analysis.

Procedure B for the Preparation of Activated Subunits:

To a solution of POCl₃ (1.1 eq) in dichloromethane was added2,6-lutidine (2 eq), followed by dropwise addition of Mo(Tr)T (7) (1eq)/dichloromethane solution at 0° C. After 1 hour, the reactionsolution was diluted with dichloromethane, and quickly washed threetimes with 10% aqueous citric acid. The desired phosphodichloridate (9)was obtained after drying over MgSO₄ and evaporation of solvent.

To a solution of the phosphodichloridate (1 eq) in dichloromethane wasadded amine (1 eq)/dichloromethane dropwise to the solution at 0° C.After 15 minutes, the reaction mixture was allowed to warm to roomtemperature for about an hour. Upon reaction completion, the product (8)as a white solid was collected by precipitation with the addition ofhexanes, followed by filtration. The product was stored at −20° C. afterdrying under vacuum. The structure was confirmed by LCMS analysis.

Example 1:((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methylphosphorodichloridate

To a cooled (ice/water bath) DCM solution (20 mL) of phosphorusoxychloride (2.12 mL, 22.7 mmol) was added dropwise 2,6-lutidine (4.82mL, 41.4 mmol) then a DCM solution (20 mL) Mo(Tr)T (2) (10.0 g, 20.7mmol) was added dropwise over 15 min (int. temp. 0-10° C.) then bath wasremoved a stirring continued at ambient temperature for 20 min. Thereaction was washed with citric acid solution (40 mL×3, 10% w/v aq),dried (MgSO₄), filtered and concentrated to a white foam (9.79 g) thenused directly for the following procedure.

Example 2:(6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl(4-(dimethylamino)piperidin-1-yl)phosphonochloridate

To a cooled (ice/water bath) DCM solution (5 mL) of thedichlorophosphate from example 1 (5.00 g, 5.00 mmol) was added a DCMsolution (5 mL) of the piperidine (0.61 g, 4.76 mmol) dropwise then thebath was removed and stirring continued at ambient temperature for 30min. The reaction was loaded directly onto a column. Chromatography with[SiO₂ column (40 g), DCM/EtOH eluant (gradient 1:0 to 1:1)] afforded thetitle compound (2.5 g) as a white foam. ESI/MS calcd. for1-(4-nitrophenyl)piperazine derivative C₄₆H₅₅N₈O₇P 862.4, foundm/z=863.6 (M+1).

Example 3:1-(1-(chloro((6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)phosphoryl)piperidin-4-yl)-1-methylpyrrolidin-1-iumchloride

The title compound was synthesized in a manner analogous to thatdescribed in Example 2 to afford the title compound (0.6 g) as a whitesolid. ESI/MS calcd. for 1-(4-nitrophenyl)piperazine derivativeC₄₉H₆₀N₈O₇P 903.4, found m/z=903.7 (M+).

Example 4:((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl(4-methylpiperazin-1-yl)phosphonochloridate

To a cooled (ice/water bath) DCM solution (10 mL) of phosphorusoxychloride (1.02 mL, 11.0 mmol) was added dropwise 2,6-lutidine (3.49mL, 29.9 mmol) then a DCM solution (10 mL) of methyl piperazine (1.00 g,10.0 mmol) was added dropwise and stirring continued for 1 h. A DCMsolution (10 mL) of Mo(Tr)T (2) (4.82, 10.0 mmol) and NMI (79 μL, 1.0mmol) was added and stirred 4 h then loaded directly onto a column.Chromatography with [SiO₂ column (80 g), DCM/Acetone with 2% TEA eluant(gradient 1:0 to 0:1)] afforded the title compound (0.8 g) as a whitefoam. ESI/MS calcd. for 1-(4-nitrophenyl)piperazine derivativeC₄₃H₄₈N₇O₈P 834.4, found m/z=835.5 (M+1).

Example 5:((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl(4-ethylpiperazin-1-yl)phosphonochloridate

The title compound was synthesized in a manner analogous to thatdescribed in Example 4 to afford the title compound (11.5 g) as a whitefoam. ESI/MS calcd. for 1-(4-nitrophenyl)piperazine derivativeC₄₅H₅₃N₈O₇P 848.4, found m/z=849.7 (M+1).

Example 6:((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl(4-ethylpiperazin-1-yl)phosphonochloridate

The title compound was synthesized in a manner analogous to thatdescribed in Example 4 to afford the title compound (4.5 g) as a whitefoam. ESI/MS calcd. for 1-(4-nitrophenyl)piperazine derivativeC₅₂H₅₆N₁₁O₆P 961.4, found m/z=962.8 (M+1).

Example 7:((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl(4-isopropylpiperazin-1-yl)phosphonochloridate

The title compound was synthesized in a manner analogous to thatdescribed in Example 4 to afford the title compound (3.5 g) as a whitefoam. ESI/MS calcd. for 1-(4-nitrophenyl)piperazine derivativeC₄₆H₅₅N₈O₇P 862.4, found m/z=863.7 (M+1).

Example 8:((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methylmethyl(2-(2,2,2-trifluoroacetamido)ethyl)phosphoramidochloridate

The title compound was synthesized in a manner analogous to thatdescribed in Example 4 to afford the title compound (1.0 g) as a whitefoam. ESI/MS calcd. for 1-(4-nitrophenyl)piperazine derivativeC₄₄H₄₈F₃N₈O₈P 904.3, found m/z=903.7 (M−1).

Example 9:((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methylmethyl(2-(2,2,2-trifluoro-N-methylacetamido)ethyl)phosphoramidochloridate

The title compound was synthesized in a manner analogous to thatdescribed in Example 4 to afford the title compound (1.8 g) as a whitefoam. ESI/MS calcd. for 1-(4-nitrophenyl)piperazine derivativeC₄₅H₅₀F₃N₈O₈P 918.3, found m/z=1836.6 (2M+).

Example 10:((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl(4-(2,2,2-trifluoroacetamido)piperidin-1-yl)phosphonochloridate

To a cooled solution (ice/water bath) of phosphorus oxychloride (17.7mL, 190 mmol) in DCM (190 mL) was added dropwise 2,6-lutidine (101 mL,864 mmol) then Mo(Tr)T (2) (83.5 g, 173 mmol) portionwise over 15 min(int. temp. 0-10° C.) and stirred. After 30 min, the 4-aminopiperidinemonotrifluoroacetamide (48.9 g, ˜190 mmol) was added dropwise over 15min (int. temp. 0-8° C.) and stirred. After 1 h, DIPEA (50 mL) was addeddropwise (int. temp. 0-10° C.) and stirred 1 h. The reaction was washedwith citric acid solution (500 mL×3, 10% w/v aq), dried (MgSO₄),filtered and concentrated to a viscous oil which was loaded directlyonto a column. Chromatography with [SiO₂ column (330 g), hexanes/EtOAceluant (gradient 1:0 to 0:1)] afforded the title compound (91.3 g, 70%yield) as a white foam. ESI/MS calcd. for 1-(4-nitrophenyl)piperazinederivative C₄₃H₄₈N₇O₈P 930.9, found m/z=954.4 (M+Na).

Examples 13-37 were prepared via procedure A described above.

Example 11:(6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl(4-(1-(2,2,2-trifluoroacetyl)piperidin-4-yl)piperazin-1-yl)phosphonochloridate

Example 12:(6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl(4-morpholinopiperidin-1-yl)phosphonochloridate

Example 13:(6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methylbis(3-(2,2,2-trifluoroacetamido)propyl)phosphoramidochloridate

Example 14:(6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl[1,4′-bipiperidin]-1′-ylphosphonochloridate

Examples 15 through 20 below were prepared via procedure B describedabove.

Example 15:(6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl(4-(pyrimidin-2-yl)piperazin-1-yl)phosphonochloridate

Example 16:(6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl(4-(2-(dimethylamino)ethyl)piperazin-1-yl)phosphonochloridate

Example 17:(6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl(4-phenylpiperazin-1-yl)phosphonochloridate

Example 18:(6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl(4-(2,2,2-trifluoro-N-methylacetamido)piperidin-1-yl)phosphonochloridate

Example 19:(6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methylmethyl(3-(2,2,2-trifluoro-N-methylacetamido)propyl)phosphoramidochloridate

Example 20:((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl(4-(2,2,2-trifluoroacetamido)piperidin-1-yl)phosphonochloridate

Example 21: (4-(pyrrolidin-1-yl)piperidin-1-yl)phosphonic dichloridehydrochloride

To a cooled (ice/water bath) solution of phosphorus oxychloride (5.70mL, 55.6 mmol) in DCM (30 mL) was added 2,6-lutidine (19.4 mL, 167 mmol)and a DCM solution (30 mL) of 4-(1-pyrrolidinyl)-piperidine (8.58 g,55.6 mmol) and stirred for 1 hour. The suspension was filtered and solidwashed with excess diethyl ether to afford the title pyrrolidine (17.7g, 91% yield) as a white solid. ESI/MS calcd. for1-(4-nitrophenyl)piperazine derivative C₁₉H₃₀N₅O₄P 423.2, foundm/z=422.2 (M−1).

Example 22:((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl(4-(pyrrolidin-1-yl)piperidin-1-yl)phosphonochloridate hydrochloride

To a stirred, cooled (ice/water bath) solution of thedichlorophosphoramidate from Example 21 (17.7 g, 50.6 mmol) in DCM (100mL) was added a DCM solution (100 mL) of Mo(Tr)T (2) (24.5 g, 50.6mmol), 2,6-Lutidine (17.7 mL, 152 mmol), and 1-methylimidazole (0.401mL, 5.06 mmol) dropwise over 10 minutes. The bath was allowed to warm toambient temperature as suspension was stirred. After 6 hours, thesuspension was poured onto diethyl ether (1 L), stirred 15 minutes,filtered and solid washed with additional ether to afford a white solid(45.4 g). The crude product was purified by chromatography [SiO₂ column(120 gram), DCM/MeOH eluant (gradient 1:0 to 6:4)], and the combinedfractions were poured onto diethyl ether (2.5 L), stirred 15 min,filtered, and the resulting solid washed with additional ether to affordthe title compound (23.1 g, 60% yield) as a white solid. ESI/MS calcd.for 1-(4-nitrophenyl)piperazine derivative C₄₈H₅₇N₈O₇P 888.4, foundm/z=887.6 (M−1).

Example 23: Preparation of Morpholino Oligomers

Preparation of trityl piperazine phenyl carbamate 1b (see FIG. 1): To acooled suspension of compound 1a in dichloromethane (6 mL/g 11) wasadded a solution of potassium carbonate (3.2 eq) in water (4 mL/gpotassium carbonate). To this two-phase mixture was slowly added asolution of phenyl chloroformate (1.03 eq) in dichloromethane (2 g/gphenyl chloroformate). The reaction mixture was warmed to 20° C. Uponreaction completion (1-2 hr), the layers were separated. The organiclayer was washed with water, and dried over anhydrous potassiumcarbonate. The product 1b was isolated by crystallization fromacetonitrile. Yield=80%.

Preparation of carbamate alcohol 1c: Sodium hydride (1.2 eq) wassuspended in 1-methyl-2-pyrrolidinone (32 mL/g sodium hydride). To thissuspension were added triethylene glycol (10.0 eq) and compound 1b (1.0eq). The resulting slurry was heated to 95° C. Upon reaction completion(1-2 hr), the mixture was cooled to 20° C. To this mixture was added 30%dichloromethane/methyl tert-butyl ether (v:v) and water. Theproduct-containing organic layer was washed successively with aqueousNaOH, aqueous succinic acid, and saturated aqueous sodium chloride. Theproduct 1c was isolated by crystallization from dichloromethane/methyltert-butyl ether/heptane. Yield=90%.

Preparation of Tail acid 1d: To a solution of compound 1c intetrahydrofuran (7 mL/g 36) was added succinic anhydride (2.0 eq) andDMAP (0.5 eq). The mixture was heated to 50° C. Upon reaction completion(5 hr), the mixture was cooled to 20° C. and adjusted to pH 8.5 withaqueous NaHCO3. Methyl tert-butyl ether was added, and the product wasextracted into the aqueous layer. Dichloromethane was added, and themixture was adjusted to pH 3 with aqueous citric acid. Theproduct-containing organic layer was washed with a mixture of pH=3citrate buffer and saturated aqueous sodium chloride. Thisdichloromethane solution of 1d was used without isolation in thepreparation of compound 1e.

Preparation of 1e: To the solution of compound 1d was addedN-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB) (1.02 eq),4-dimethylaminopyridine (DMAP) (0.34 eq), and then1-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (1.1eq). The mixture was heated to 55° C. Upon reaction completion (4-5 hr),the mixture was cooled to 20° C. and washed successively with 1:1 0.2 Mcitric acid/brine and brine. The dichloromethane solution underwentsolvent exchange to acetone and then to N,N-dimethylformamide, and theproduct was isolated by precipitation from acetone/N,N-dimethylformamideinto saturated aqueous sodium chloride. The crude product was reslurriedseveral times in water to remove residual N,N-dimethylformamide andsalts. Yield=70% of 1e from compound 1c. Introduction of the activated“Tail” onto the disulfide anchor-resin was performed in NMP by theprocedure used for incorporation of the subunits during solid phasesynthesis.

Preparation of the Solid Support for Synthesis of Morpholino Oligomers(see FIG. 2): This procedure was performed in a silanized, jacketedpeptide vessel (custom made by ChemGlass, NJ, USA) with a coarseporosity (40-60 μm) glass frit, overhead stirrer, and 3-way Teflonstopcock to allow N2 to bubble up through the frit or a vacuumextraction. Temperature control was achieved in the reaction vessel by acirculating water bath.

The resin treatment/wash steps in the following procedure consist of twobasic operations: resin fluidization and solvent/solution extraction.For resin fluidization, the stopcock was positioned to allow N2 flow upthrough the frit and the specified resin treatment/wash was added to thereactor and allowed to permeate and completely wet the resin. Mixing wasthen started and the resin slurry mixed for the specified time. Forsolvent/solution extraction, mixing and N2 flow were stopped and thevacuum pump was started and then the stopcock was positioned to allowevacuation of resin treatment/wash to waste. All resin treatment/washvolumes were 15 mL/g of resin unless noted otherwise.

To aminomethylpolystyrene resin (100-200 mesh; ˜1.0 mmol/g N2substitution; 75 g, 1 eq, Polymer Labs, UK, part #1464-X799) in asilanized, jacketed peptide vessel was added 1-methyl-2-pyrrolidinone(NMP; 20 ml/g resin) and the resin was allowed to swell with mixing for1-2 hr. Following evacuation of the swell solvent, the resin was washedwith dichloromethane (2×1-2 min), 5% diisopropylethylamine in 25%isopropanol/dichloromethane (2×3-4 min) and dichloromethane (2×1-2 min).After evacuation of the final wash, the resin was fluidized with asolution of disulfide anchor 2a in 1-methyl-2-pyrrolidinone (0.17 M; 15mL/g resin, ˜2.5 eq) and the resin/reagent mixture was heated at 45° C.for 60 hr. On reaction completion, heating was discontinued and theanchor solution was evacuated and the resin washed with1-methyl-2-pyrrolidinone (4×3-4 min) and dichloromethane (6×1-2 min).The resin was treated with a solution of 10% (v/v) diethyl dicarbonatein dichloromethane (16 mL/g; 2×5-6 min) and then washed withdichloromethane (6×1-2 min). The resin 2b was dried under a N2 streamfor 1-3 hr and then under vacuum to constant weight (±2%). Yield:110-150% of the original resin weight.

Determination of the Loading of Aminomethylpolystyrene-disulfide resin:The loading of the resin (number of potentially available reactivesites) is determined by a spectrometric assay for the number oftriphenylmethyl (trityl) groups per gram of resin.

A known weight of dried resin (25±3 mg) is transferred to a silanized 25ml volumetric flask and ˜5 mL of 2% (v/v) trifluoroacetic acid indichloromethane is added. The contents are mixed by gentle swirling andthen allowed to stand for 30 min. The volume is brought up to 25 mL withadditional 2% (v/v) trifluoroacetic acid in dichloromethane and thecontents thoroughly mixed. Using a positive displacement pipette, analiquot of the trityl-containing solution (500 μL) is transferred to a10 mL volumetric flask and the volume brought up to 10 mL withmethanesulfonic acid.

The trityl cation content in the final solution is measured by UVabsorbance at 431.7 nm and the resin loading calculated in trityl groupsper gram resin (μmol/g) using the appropriate volumes, dilutions,extinction coefficient (ε: 41 μmol-1 cm⁻¹) and resin weight. The assayis performed in triplicate and an average loading calculated.

The resin loading procedure in this example will provide resin with aloading of approximately 500 μmol/g. A loading of 300-400 in μmol/g wasobtained if the disulfide anchor incorporation step is performed for 24hr at room temperature.

Tail loading: Using the same setup and volumes as for the preparation ofaminomethylpolystyrene-disulfide resin, the Tail can be introduced intothe molecule. For the coupling step, a solution of 1e (0.2 M) in NMPcontaining 4-ethylmorpholine (NEM, 0.4 M) was used instead of thedisulfide anchor solution. After 2 hr at 45° C., the resin 2b was washedtwice with 5% diisopropylethylamine in 25% isopropanol/dichloromethaneand once with DCM. To the resin was added a solution of benzoicanhydride (0.4 M) and NEM (0.4 M). After 25 min, the reactor jacket wascooled to room temperature, and the resin washed twice with 5%diisopropylethylamine in 25% isopropanol/dichloromethane and eight timeswith DCM. The resin 2c was filtered and dried under high vacuum. Theloading for resin 2c is defined to be the loading of the originalaminomethylpolystyrene-disulfide resin 2b used in the Tail loading.

Solid Phase Synthesis: Morpholino Oligomers were prepared on a GilsonAMS-422 Automated Peptide Synthesizer in 2 mL Gilson polypropylenereaction columns (Part #3980270). An aluminum block with channels forwater flow was placed around the columns as they sat on the synthesizer.The AMS-422 will alternatively add reagent/wash solutions, hold for aspecified time, and evacuate the columns using vacuum.

For oligomers in the range up to about 25 subunits in length,aminomethylpolystyrene-disulfide resin with loading near 500 μmol/g ofresin is preferred. For larger oligomers,aminomethylpolystyrene-disulfide resin with loading of 300-400 μmol/g ofresin is preferred. If a molecule with 5′-Tail is desired, resin thathas been loaded with Tail is chosen with the same loading guidelines.

The following reagent solutions were prepared:

Detritylation Solution: 10% Cyanoacetic Acid (w/v) in 4:1dichloromethane/acetonitrile; Neutralization Solution: 5%Diisopropylethylamine in 3:1 dichloromethane/isopropanol; CouplingSolution: 0.18 M (or 0.24 M for oligomers having grown longer than 20subunits) activated Morpholino Subunit of the desired base and linkagetype and 0.4 M N ethylmorpholine, in 1,3-dimethylimidazolidinone.Dichloromethane (DCM) was used as a transitional wash separating thedifferent reagent solution washes.

On the synthesizer, with the block set to 42° C., to each columncontaining 30 mg of aminomethylpolystyrene-disulfide resin (or Tailresin) was added 2 mL of 1-methyl-2-pyrrolidinone and allowed to sit atroom temperature for 30 min. After washing with 2 times 2 mL ofdichloromethane, the following synthesis cycle was employed:

Step Volume Delivery Hold time Detritylation 1.5 mL Manifold 15 secondsDetritylation 1.5 mL Manifold 15 seconds Detritylation 1.5 mL Manifold15 seconds Detritylation 1.5 mL Manifold 15 seconds Detritylation 1.5 mLManifold 15 seconds Detritylation 1.5 mL Manifold 15 secondsDetritylation 1.5 mL Manifold 15 seconds DCM 1.5 mL Manifold 30 secondsNeutralization 1.5 mL Manifold 30 seconds Neutralization 1.5 mL Manifold30 seconds Neutralization 1.5 mL Manifold 30 seconds Neutralization 1.5mL Manifold 30 seconds Neutralization 1.5 mL Manifold 30 secondsNeutralization 1.5 mL Manifold 30 seconds DCM 1.5 mL Manifold 30 secondsCoupling 350 uL-500 uL Syringe 40 minutes DCM 1.5 mL Manifold 30 secondsNeutralization 1.5 mL Manifold 30 seconds Neutralization 1.5 mL Manifold30 seconds DCM 1.5 mL Manifold 30 seconds DCM 1.5 mL Manifold 30 secondsDCM 1.5 mL Manifold 30 seconds

The sequences of the individual oligomers were programmed into thesynthesizer so that each column receives the proper coupling solution(A, C, G, T, I) in the proper sequence. When the oligomer in a columnhad completed incorporation of its final subunit, the column was removedfrom the block and a final cycle performed manually with a couplingsolution comprised of 4-methoxytriphenylmethyl chloride (0.32 M in DMI)containing 0.89 M 4-ethylmorpholine.

Cleavage from the resin and removal of bases and backbone protectinggroups: After methoxytritylation, the resin was washed 8 times with 2 mL1-methyl-2-pyrrolidinone. One mL of a cleavage solution consisting of0.1 M 1,4-dithiothreitol (DTT) and 0.73 M triethylamine in1-methyl-2-pyrrolidinone was added, the column capped, and allowed tosit at room temperature for 30 min. After that time, the solution wasdrained into a 12 mL Wheaton vial. The greatly shrunken resin was washedtwice with 300 μL of cleavage solution. To the solution was added 4.0 mLconc aqueous ammonia (stored at −20° C.), the vial capped tightly (withTeflon lined screw cap), and the mixture swirled to mix the solution.The vial was placed in a 45° C. oven for 16-24 hr to effect cleavage ofbase and backbone protecting groups.

Initial Oligomer Isolation: The vialed ammonolysis solution was removedfrom the oven and allowed to cool to room temperature. The solution wasdiluted with 20 mL of 0.28% aqueous ammonia and passed through a 2.5×10cm column containing Macroprep HQ resin (BioRad). A salt gradient (A:0.28% ammonia with B: 1 M sodium chloride in 0.28% ammonia; 0-100% B in60 min) was used to elute the methoxytrityl containing peak. Thecombined fractions were pooled and further processed depending on thedesired product.

Demethoxytritylation of Morpholino Oligomers: The pooled fractions fromthe Macroprep purification were treated with 1 M H3PO4 to lower the pHto 2.5. After initial mixing, the samples sat at room temperature for 4min, at which time they are neutralized to pH 10-11 with 2.8%ammonia/water. The products were purified by solid phase extraction(SPE).

Amberchrome CG-300M (Rohm and Haas; Philadelphia, Pa.) (3 mL) is packedinto 20 mL fritted columns (BioRad Econo-Pac Chromatography Columns(732-1011)) and the resin rinsed with 3 mL of the following: 0.28%NH4OH/80% acetonitrile; 0.5M NaOH/20% ethanol; water; 50 mM H3PO4/80%acetonitrile; water; 0.5 NaOH/20% ethanol; water; 0.28% NH4OH.

The solution from the demethoxytritylation was loaded onto the columnand the resin rinsed three times with 3-6 mL 0.28% aqueous ammonia. AWheaton vial (12 mL) was placed under the column and the product elutedby two washes with 2 mL of 45% acetonitrile in 0.28% aqueous ammonia.The solutions were frozen in dry ice and the vials placed in a freezedryer to produce a fluffy white powder. The samples were dissolved inwater, filtered through a 0.22 micron filter (Pall Life Sciences,Acrodisc 25 mm syringe filter, with a 0.2 micron HT Tuffryn membrane)using a syringe and the Optical Density (OD) was measured on a UVspectrophotometer to determine the OD units of oligomer present, as wellas dispense sample for analysis. The solutions were then placed back inWheaton vials for lyophilization.

Analysis of Morpholino Oligomers: MALDI-TOF mass spectrometry was usedto determine the composition of fractions in purifications as well asprovide evidence for identity (molecular weight) of the oligomers.Samples were run following dilution with solution of3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid),3,4,5-trihydroxyacetophenone (THAP) or alpha-cyano-4-hydroxycinnamicacid (HCCA) as matrices.

Cation exchange (SCX) HPLC was performed using a Dionex ProPac SCX-10,4×250 mm column (Dionex Corporation; Sunnyvale, Calif.) using 25 mM pH=5sodium acetate 25% acetonitrile (Buffer A) and 25 mM pH=5 sodium acetate25% acetonitrile 1.5 M potassium chloride (buffer B) (Gradient 10-100% Bin 15 min) or 25 mM KH2PO4 25% acetonitrile at pH=3.5 (buffer A) and 25mM KH2PO4 25% acetonitrile at pH=3.5 with 1.5 M potassium chloride(buffer B) (Gradient 0-35% B in 15 min). The former system was used forpositively charged oligomers that do not have a peptide attached, whilethe latter was used for peptide conjugates.

Purification of Morpholino Oligomers by Cation Exchange Chromatography:The sample is dissolved in 20 mM sodium acetate, pH=4.5 (buffer A) andapplied to a column of Source 30 cation exchange resin (GE Healthcare)and eluted with a gradient of 0.5 M sodium chloride in 20 mM sodiumacetate and 40% acetonitrile, pH=4.5 (buffer B). The pooled fractionscontaining product are neutralized with conc aqueous ammonia and appliedto an Amberchrome SPE column. The product is eluted, frozen, andlyophilized as above.

Example 24: APN Oligonucleotide Modification Enhances SMN2 Exon 7Inclusion

Oligonucleotides containing the sequences shown in FIG. 5 were tested todetermine whether the APN oligonucleotide would enhance SMN2 Exon 7inclusion. Each of the oligonucleotides shown in Table 1 were introducedinto cells using the Nucleofection Protocol described below. The resultswere quantified using the reverse transcriptase protocol and are shownin FIGS. 6 and 7. Intensity of the gel bands representing inclusion orexclusion of SMN2 exon 7 in GM03813 fibroblast cells (Coriell) werequantified with ImageQuant (GE). Exon 7 inclusion is reported as apercentage calculated from the ratio of the exon 7-included bandintensities divided by the sum of the intensities from the exon7-included and -excluded bands. Each dot represents the mean+/−1standard deviation of two replicates at each concentration. Threeindependent experiments were combined to yield the above dataset.Percent inclusion analysis was performed in Microsoft Excel. Data pointsand curves were plotted in Graphpad Prism. As shown in FIG. 6 theaddition of apn modifications to an oligonucleotide enhances the potencyof the compound compared to unmodified PMO containing the same sequence.Thus, as shown in FIG. 6, 14 mer (11/15) APN was about an order ofmagnitude more potent than 14 mer (11/15) without the APN linkage.Likewise E8/4a-APN (11/15) and E8/4b-APN (11/15) were more potent thanE8/4a (11/15) and E8/4b (11/15) respectively.

Example 25: SMA Cells Nucleofection Protocol

Patient-derived fibroblasts from an individual with Spinal MuscularAtrophy (Coriell cell line GM03813) were cultured according to standardprotocols in Eagle's MEM with 10% FBS. Cells were passaged 3-5 daysbefore the experiment and were approximately 80% confluent atnucleofection. Oligos were prepared as 1-2 mM stock solutions innuclease-free water (not treated with DEPC) from which appropriatedilutions were made for nucleofection. Fibroblasts were trypsinized,counted, centrifuged at 90 g for 10 minutes, and 1-5×10e5 cells per wellwere resuspended in nucleofection Solution P2 (Lonza). Oligo solutionand cells were then added to each well of a Nucleocuvette 16-well strip,and pulsed with program EN-100. Cells were incubated at room temperaturefor 10 minutes and transferred to a 12-well plate in duplicate. TotalRNA was isolated from treated cells after 48 hours using the GE Illustra96 Spin kit following the manufacturer's recommended protocol. RecoveredRNA was stored at −80° C. prior to analysis.

Reverse transcriptase PCR was performed to amplify the SMN2 allele usingthe SuperScript III One-Step RT-PCR system (Invitrogen). 400 ng totalRNA isolated from nucleofected cells was reverse transcribed andamplified with the following gene-specific primers and conditions(described in Hua 2007): E6-F: 5′ ATA ATT CCC CCA CCA CCT CCC 3′ (SEQ IDNO: 80); E8-467-R: 5′ TTG CCA CAT ACG CCT CAC ATA C 3′ (SEQ ID NO: 81);PCR Program: 60° C. for 30 min RT incubation; 94° C. denature, 55° C.anneal, 72° C. extension, 22 cycles. The amplification solution providedin the One-Step kit was supplemented with Cy5-labeled dCTP (GE) toenable band visualization by fluorescence. Following amplification, PCRproducts were digested with DDEI to differentiate between SMN1 or SMN2alleles (as described in Hua 2007). Digested samples were run on apre-cast 10% acrylamide/TBE gel (Invitrogen) and visualized on a TyphoonTrio (GE) using the 633 nm excitation laser and 670 nm BP 30 emissionfilter with the focal plane at the platen surface. Gels were analyzedwith ImageQuant (GE) to determine the intensities of the bands.Intensities from all bands containing exon 7 were added together torepresent the full exon 7 transcript levels in the inclusion analysis.

Example 26: SMA Mouse Model

SMNΔ7 mice can be used as an SMA model to characterize disease modifyingantisense oligonucleotides. Mice possess only one Smn gene and the lossof this gene is embryonic lethal. To generate mice with an SMNdeficiency that models human SMA, the human SMN2 gene can be introducedinto mice. For example, two copies of human SMN2 can be introduced intomice lacking Smn to generate mice with severe SMA that may live anaverage of 5 days, while eight copies of SMN2 can be introduced torescue the mice. In addition, SMN17, a SMN transgene lacking exon 7, canbe introduced into the severe SMA mice to increase the average lifespan. Furthermore, SMN can be induced postnatally to modulate SMA inSMNΔ7 mice. Thus, the SMNΔ7 mouse can be used as an SMA model tocharacterize disease modifying antisense oligonucleotides.

Using the SMNΔ7 mouse model of SMA, multiple treatment strategies toincrease SMN expression can be performed. Targeting SMN production withvarious pharmacologic compounds, such as antisense oligomers, to eitheractivate the SMN promoter or to alter exon 7-splicing patterns can beperformed to improve the phenotype of SMNΔ7 SMA mice. For example,antisense oligonucleotides can block target sequences, including exonsplice enhancers or intron splice silencers (ISSs). Furthermore, SMN canbe induced postnatally to achieve a therapeutic effect.

Antisense oligomers such as PMO, PMO+, PPMO, and PMO-X, can be deliveredto mice via ICV injection at high concentration to alter SMN2 splicingand increase SMN levels, Treated SMA mice may demonstrate improvement inweight gain, motor activity and increased survival time. Antisenseoligomers may be delivered by several mechanisms including, but notlimited to intracerebroventriclar (ICV) injection, peripheral FVdelivery, combined peripheral and ICV delivery and dual ICV injection.

Early CNS treatment will likely yield robust effects. However, delayedCNS delivery may still increase survival levels.

ICV injection may yield an increase in both SMN2 exon 7 incorporationand SMN protein levels in brain and spinal cord. Thus, it may bepreferable to restore SMN levels within neurons to have an impact inSMA. It may be possible that long survival benefit can be obtainedwithout significantly enhancing SMN levels in the periphery. However, itis also possible that increasing SMN expression in the autonomic nervoussystem outside of the blood-brain barrier results in correction of SMA.

TABLE 1 PMO and APN-modified sequences for SMA-targeted oligonucleotides.APN modified positions are identified in bold and underlined. Sequence SampleName Sequence Identifier N1 ATT CAC TTT CAT AAT GCT GGSEQ ID NO: 1 N1-B AT T  CAC TTT CAT AA T  GC T  GG SEQ ID NO: 2 N1-CATT CAC T T T CA T  AA T  GCT GG SEQ ID NO: 3 N1-D AT T  CAC T TT CAT AA T  GC T  GG SEQ ID NO: 4 N1-E AT T  CAC T T T CA T  AA T  GC T GG SEQ ID NO: 5 N1-F AT T  CAC T T T CA T  AA T  GC T  GG SEQ ID NO: 6N1-G AT T  C A C T T T CA T  AA T  GC T  GG SEQ ID NO: 7 N1-H AT T  C AC T T T  C A T  AA T  GC T  GG SEQ ID NO: 8 AVI-17merCAC TTT CAT AAT GCT GG SEQ ID NO: 9 AVI-17mer-B CAC  T TT CAT AA T  GC T GG SEQ ID NO: 10 AVI-17mer-C CAC  T TT CA T  AA T  GC T  GGSEQ ID NO: 11 AN/-17mer-D CAC  T T T  CA T  AA T  GC T  GG SEQ ID NO: 12AVI-17mer-E C A C  T T T  CA T  AA T  GC T  GG SEQ ID NO: 13 AVI-17mer-FC A C  T T T  CA T   A A T  GC T  GG SEQ ID NO: 14 AVI-17mer-G C A C  TT T   C A T   A A T  GC T  GG SEQ ID NO: 15 AVI-17mer-H C A C  T T T   CA T   A A T   G C T  GG SEQ ID NO: 16 AVI-17mer-I CAC TTT C A T  A A T GCT GG SEQ ID NO: 17 AVI-17mer-J C A C  T TT CAT AAT GC T  GGSEQ ID NO: 18 14mer TTT CAT AAT GCT GG SEQ ID NO: 19 14mer-B T T T CA T AAT GC T  GG SEQ ID NO: 20 14mer-APN T T T CA T  AA T  GC T  GGSEQ ID NO: 21 14mer-C T T T CA T   A A T  GC T  GG SEQ ID NO: 22 14mer-DT T T  C A T   A A T  GC T  GG SEQ ID NO: 23 14mer-E T T T  C A T   A AT   G C T  GG SEQ ID NO: 24 14mer-F TTT  C A T   A A T  GC T   G GSEQ ID NO: 25 3UP11 AAT GCT GGC AG SEQ ID NO: 26 11mer-APN AA T  GC T GGC AG SEQ ID NO: 27 11mer-B AA T  GC T  GGC  A G SEQ ID NO: 28 11mer-CAA T  GC T  GG C   A G SEQ ID NO: 29 11mer-D AA T  G CT  GG C   A GSEQ ID NO: 30 11mer-E AA T  G CT   G G C   A G SEQ ID NO: 31 11mer-F A AT  G CT   G G C   A G SEQ ID NO: 32 3UP8 GCT GGC AG SEQ ID NO: 338mer-APN GC T  GGC AG SEQ ID NO: 34 8mer-B GC T  GGC  A G SEQ ID NO: 358mer-C GCT  G G C  AG SEQ ID NO: 36 8mer-D GC T   G G C  AGSEQ ID NO: 37 8mer-E GC T  GG C   A G SEQ ID NO: 38 8mer-F G CT  GG C  A G SEQ ID N0: 39 8mer-G G CT   G G C   A G SEQ ID NO: 40 8mer-H G C T  G G C   A G SEQ ID NO: 41 E8/4a C TAG TAT TTC CTG CAA ATG AGSEQ ID NO: 42 E8/4a-APN C  T AG  T AT TTC C T G CAA A T G AGSEQ ID NO: 43 E8/4a-B C  T AG  T AT  T TC C T G CAA A T G AGSEQ ID NO: 44 E8/4a-C C  T AG  T AT  T TC C T G C A A A T G AGSEQ ID NO: 45 E8/4a-D C  T AG  T AT  T TC C T G C A A A T G  A GSEQ ID NO: 46 E8/4a-E C   T AG  T AT  T TC C T G C A A A T G AGSEQ ID NO: 47 E8/4a-F C  T AG  T AT TTC CTG CAA A T G  A G SEQ ID NO: 48E8/4a-G C  T AG  T AT TTC CTG CAA A T G AG SEQ ID NO: 49 E8/4a-HC TAG TAT T T C C T G CAA A T G AG SEQ ID NO: 50 E8/4a-I C TAG TAT T TC C T G C A A ATG AG SEQ ID NO: 51 E8/4a-J C TAG TA T  T T C C T G C AA ATG AG SEQ ID NO: 52 E8/4b C CAG CAT TTC CTG CAA ATG AG SEQ ID NO: 53E8/4b-APN C CAG CA T  T T C C T G CAA A T G AG SEQ ID NO: 54 E8/4b-BC CAG CA T  T T C C T G C A A A T G AG SEQ ID NO: 55 E8/4b-C C C A G CAT  T T C  C TG C A A A T G AG SEQ ID NO: 56 E8/4b-D C CA G  CA T  T T C C TG C A A A T G AG SEQ ID NO: 57 E8/4b-E C C A G CA T  TTC CTG CAA A TG AG SEQ ID NO: 58 E8/4b-F C C A G CA T  TTC CTG CAA A T G  A GSEQ ID NO: 59 E8/4b-G C CAG CAT T T C C T G C A A ATG AG SEQ ID NO: 60E8/4b-H C CAG CAT T T C C T G C A A  A TG AG SEQ ID NO: 61 E8/3A TGC CAG CAT TTC CTG CAA ATG AGA SEQ ID NO: 62 E8/3-B A  T GC CAG CA T TTC C T G CAA A T G AGA SEQ ID NO: 63 E8/3-C A  T GC C AG CAT TTC CTG C A A A T G AGA SEQ ID NO: 64 E8/3-D A  T GC C A G CA T TTC C T G C A A A T G AGA SEQ ID NO: 65 E8/3-E A  T GC C A G  CAT TTC C T G  C AA A T G AGA SEQ ID NO: 66 E8/3-F A TGC C A G CAT  TTC C T G  C AA ATG A G A SEQ ID NO: 67 E8/3-G A  T GC CAG CAT TTC C TG CAA A T G AGA SEQ ID NO: 68 E8/3-H A  T GC CAG CAT TTC CTG CAA A TG AGA SEQ ID NO: 69 E8/4 GCT CTA TGC CAG CAT TTC CTG CAA A SEQ ID NO: 70E8/4-B GCT C T A TGC CAG CAT TTC C T G CAA A SEQ ID NO: 71 E8/4-C GCT CT A TGC CAG CA T  TTC C T G CAA A SEQ ID NO: 72 E8/4-D GCT C T A TGC C AG CA T  TTC C T G CAA A SEQ ID NO: 73 E8/4-E GC T  C TA TGC CAG CAT TTC C T G C A A A SEQ ID NO: 74 E8/4-F GCT CTA TG C  C AG C A T  T TC CTG CAA A SEQ ID NO: 75 E8/4-G GC T  C T A TGC CAG CA T TTC C T G CAA A SEQ ID NO: 76 E8/4-H GC T  C T A TGC CAG CA T  T TC CTG CAA A SEQ ID NO: 77 E8/4-I GC T  C T A TGC CAG CA T  T T C C TG CAA A SEQ ID NO: 78 E8/4-J GC T  C T A TGC CAG CA T  T T C CTG C A A ASEQ ID NO: 79

What is claimed:
 1. An antisense oligonucleotide of 10-40 nucleotides inlength which specifically hybridizes to a region within the SMN2 gene,such that the level of exon 7-containing SMN2 mRNA relative to exon7-deleted SMN2 mRNA in the cell is enhanced, wherein the antisenseoligonucleotide is a morpholino oligonucleotide, and wherein eachnucleotide is a nucleotide having a formula:

wherein Nu is a nucleobase; R₁ is a moiety of the formula (I):

or R₁ is —N(CH₃)₂; q is 0, 1, 2, 3 or 4; R₂ is selected from the groupconsisting of hydrogen, C₁-C₅ alkyl, and a formamidinyl moiety, and R₃is selected from the group consisting of hydrogen and C₁-C₅ alkyl, or R₂and R₃ are joined to form a 5-7 membered heterocyclic ring optionallycontaining an oxygen hetero atom, where the ring may be optionallysubstituted with a substituent selected from the group consisting ofC₁-C₅ alkyl, phenyl, halogen, and aralkyl; R₄ is selected from the groupconsisting of null, hydrogen, a C₁-C₆ alkyl and aralkyl; R_(x) isselected from the group consisting of HO—, a nucleotide, andpiperazinyl; R_(y) is selected from the group consisting of hydrogen, aC₁-C₆ alkyl, a nucleotide, a, an amino acid, a formamidinyl moiety, andacyl; and, R_(z) is selected from the group consisting of null,hydrogen, a C₁-C₆ alkyl, and acyl; and pharmaceutically acceptable saltsthereof, wherein at least one R₁ is of formula (I).
 2. The antisenseoligonucleotide of claim 1, wherein at least one nucleotide has theformula:

wherein Rx, Ry, Rz, and Nu are as stated in claim
 1. 3. The antisenseoligonucleotide of claim 1, wherein the target region is within exon 7,intron 7, or exon 8 of the SMN2 gene.
 4. The antisense oligonucleotideof claim 3, wherein the antisense oligonucleotide comprises a sequencewhich is complementary to a target region within intron 7 of the SMN2gene.
 5. The antisense oligonucleotide of claim 1, wherein the antisenseoligonucleotide comprises a sequence which is complementary to a portionof intron 7 and exon 8 of the SMN2 gene.
 6. The antisenseoligonucleotide of claim 1, wherein the antisense oligonucleotidecomprises a sequence of from about 10 to about 30 nucleotides.
 7. Theantisense oligonucleotide of claim 1, wherein the antisenseoligonucleotide comprises a sequence of from about 14 to about 21nucleotides.
 8. The antisense oligonucleotide of claim 1, wherein theantisense oligonucleotide comprises a sequence selected from SEQ ID NOS:2-8, 10-18, 20-25, 27-32, 34-41, 43-52, 54-61, 63-69, and 71-79.
 9. Amethod of enhancing the level of exon 7-containing SMN2 mRNA relative toexon-deleted SMN2 mRNA in a cell, comprising contacting the cell with anantisense oligonucleotide of 10-40 nucleotides in length whichspecifically hybridizes to a region within the SMN2 gene, such that thelevel of exon 7-containing SMN2 mRNA relative to exon 7-deleted SMN2mRNA in the cell is enhanced, wherein the antisense oligonucleotide is amorpholino oligonucleotide, and wherein each nucleotide is a nucleotidehaving a formula:

wherein Nu is a nucleobase; R₁ is a moiety of the formula (I):

or R₁ is —N(CH₃)₂; q is 0, 1, 2, 3 or 4; R₂ is selected from the groupconsisting of hydrogen, C₁-C₅ alkyl, and a formamidinyl moiety, and R₃is selected from the group consisting of hydrogen and C₁-C₅ alkyl, or R₂and R₃ are joined to form a 5-7 membered heterocyclic ring optionallycontaining an oxygen hetero atom, where the ring may be optionallysubstituted with a substituent selected from the group consisting ofC₁-C₅ alkyl, phenyl, halogen, and aralkyl; R₄ is selected from the groupconsisting of null, hydrogen, a C₁-C₆ alkyl and aralkyl; R_(x) isselected from the group consisting of HO—, a nucleotide, andpiperazinyl; R_(y) is selected from the group consisting of hydrogen, aC₁-C₆ alkyl, a nucleotide, a an amino acid, a formamidinyl moiety, andacyl; and, R_(z) is selected from the group consisting of null,hydrogen, a C₁-C₆ alkyl, and acyl; and pharmaceutically acceptable saltsthereof, wherein at least one R₁ is of formula (I).
 10. The method ofclaim 9, wherein at least one nucleotide has the formula:

wherein Rx, Ry, Rz, and Nu are as stated in claim
 9. 11. The method ofclaim 10, wherein Nu is thymine or uracil.
 12. The method of claim 9,wherein the target region is within exon 7, intron 7, or exon 8 of theSMN2 gene.
 13. The method of claim 12, wherein the antisenseoligonucleotide comprises a sequence which is complementary to intron 7of the SMN2 gene.
 14. The method of claim 13, wherein the antisenseoligonucleotide comprises a sequence which is complementary to a portionof intron 7 and exon 8 of the SMN2 gene.
 15. The method of claim 9,wherein the antisense oligonucleotide comprises a sequence of from about10 to about 30 nucleotides.
 16. The method of claim 9, wherein theantisense oligonucleotide comprises a sequence of from about 14 to about21 nucleotides.
 17. The method of claim 9, wherein the antisenseoligonucleotide comprises a sequence selected from SEQ ID NOS: 2-8,10-18, 20-25, 27-32, 34-41, 43-52, 54-61, 63-69, and 71-79.
 18. A methodof treating spinal muscular atrophy (SMA) in a patient, comprisingadministering to the patient an antisense oligonucleotide according toclaim 1, thereby treating the patient.